Carbamate compounds as 5-HT4 receptor agonists

ABSTRACT

The invention provides novel benzoimidazolone-carboxamide-derived carbamate 5-HT 4  receptor agonist compounds of formula (I):  
                 
 
wherein R 1 , R 2 , R 3 , R 4 , a, and b are defined in the disclosure. The invention also provides pharmaceutical compositions comprising such compounds, methods of using such compounds to treat diseases associated with 5-HT 4  receptor activity, and processes and intermediates useful for preparing such compounds.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.60/738,702 filed on Nov. 22, 2005, the disclosure of which isincorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention is directed to benzoimidazolone-carboxamide-derivedcarbamate compounds which are useful as 5-HT₄ receptor agonists. Theinvention is also directed to pharmaceutical compositions comprisingsuch compounds, methods of using such compounds for treating orpreventing medical conditions mediated by 5-HT₄ receptor activity, andprocesses and intermediates useful for preparing such compounds.

2. State of the Art

Serotonin (5-hydroxytryptamine, 5-HT) is a neurotransmitter that iswidely distributed throughout the body, both in the central nervoussystem and in peripheral systems. At least seven subtypes of serotoninreceptors have been identified and the interaction of serotonin withthese different receptors is linked to a wide variety of physiologicalfunctions. There has been, therefore, substantial interest in developingtherapeutic agents that target specific 5-HT receptor subtypes.

In particular, characterization of 5-HT₄ receptors and identification ofpharmaceutical agents that interact with them has been the focus ofsignificant recent activity. (See, for example, the review by Langloisand Fischmeister, J. Med. Chem. 2003, 46, 319-344.) 5-HT₄ receptoragonists are useful for the treatment of disorders of reduced motilityof the gastrointestinal tract. Such disorders include irritable bowelsyndrome (IBS), chronic constipation, functional dyspepsia, delayedgastric emptying, gastroesophageal reflux disease (GERD), gastroparesis,post-operative ileus, intestinal pseudo-obstruction, and drug-induceddelayed transit. In addition, it has been suggested that some 5-HT₄receptor agonist compounds may be used in the treatment of centralnervous system disorders including cognitive disorders, behavioraldisorders, mood disorders, and disorders of control of autonomicfunction.

Despite the broad utility of pharmaceutical agents modulating 5-HT₄receptor activity, few 5-HT₄ receptor agonist compounds are in clinicaluse at present.

Accordingly, there is a need for new 5-HT₄ receptor agonists thatachieve their desired effects with minimal side effects. Preferredagents may possess, among other properties, improved selectivity,potency, pharmacokinetic properties, and/or duration of action.

SUMMARY OF THE INVENTION

The invention provides novel compounds that possess 5-HT₄ receptoragonist activity. Among other properties, compounds of the inventionhave been found to be potent and selective 5-HT₄ receptor agonists. Inaddition, compounds of the invention have been found to exhibitfavorable pharmacokinetic properties which are predictive of goodbioavailability upon oral administration.

Accordingly, the invention provides a compound of formula (I):

wherein:

R¹ is halo or C₁₋₃alkyl, wherein C₁₋₃alkyl is optionally substitutedwith hydroxy or halo;

R² is hydrogen or C₁₋₃alkyl, wherein C₁₋₃alkyl is optionally substitutedwith hydroxy;

R³ is C₁₋₃alkyl or hydrogen;

R⁴ is —(CH₂)₁₋₃C(O)NR^(a)R^(b),

or R³ and R⁴ together with the nitrogen atom to which they are attachedform a moiety selected from:

-   -   (i) a moiety of formula (a):    -   (ii) a moiety of formula (b):    -   (iii) a moiety of formula (c):

wherein:

R⁵ is —OC(O)NR^(a)R^(b)), —C(O)NR^(a)R^(b), —NR^(d)S(O)₂C₁₋₃alkyl,—NR^(d)C(O)R^(c), —NR^(d)S(O)₂NR^(a)R^(b), or —NR^(d)C(O)OR^(e);

R⁶ is —C(O)R^(f), —(CH₂)₂OR^(g), —S(O)₂NR^(a)R^(b), —S(O)₂C₁₋₃alkyl, or—S(O)₂(CH₂)₁₋₃S(O)₂C₁₋₃alkyl;

R^(a), R^(b), and R^(c) are independently hydrogen or C₁₋₃alkyl;

R^(d) is hydrogen or C₁₋₃alkyl, wherein C₁₋₃alkyl is optionallysubstituted with hydroxy;

R^(e) is C₁₋₃alkyl;

R^(f) is hydrogen, C₁₋₃alkyl, tetrahydrofuranyl, or —NR^(a)R^(b);

R^(g) is hydrogen or C₁₋₃alkyl;

a is 0, 1 or 2;

b is 0, 1, 2 or 3;

c is 0, 1, or 2;

d is 1 or 2; and

e is 1 or 2;

provided that when c is 0, then d is 2, and R⁵ is —C(O)NR^(a)R^(b); andwhen c is 2, then d is 1;

or a pharmaceutically-acceptable salt or solvate or stereoisomerthereof.

The invention also provides a pharmaceutical composition comprising acompound of the invention and a pharmaceutically-acceptable carrier.

Further, the invention provides a method of treating a disease orcondition associated with 5-HT₄ receptor activity, e.g. a disorder ofreduced motility of the gastrointestinal tract, the method comprisingadministering to the mammal, a therapeutically effective amount of acompound or of a pharmaceutical composition of the invention.

The compounds of the invention can also be used as research tools, i.e.to study biological systems or samples, or for studying the activity ofother chemical compounds. Accordingly, in another of its method aspects,the invention provides a method of using a compound of formula (I), or apharmaceutically acceptable salt or solvate or stereoisomer thereof, asa research tool for studying a biological system or sample or fordiscovering new 5-HT₄ receptor agonists, the method comprisingcontacting a biological system or sample with a compound of theinvention and determining the effects caused by the compound on thebiological system or sample.

In separate and distinct aspects, the invention also provides syntheticprocesses and intermediates described herein, which are useful forpreparing compounds of the invention.

The invention also provides a compound of the invention as describedherein for use in medical therapy, as well as the use of a compound ofthe invention in the manufacture of a formulation or medicament fortreating a disease or condition associated with 5-HT₄ receptor activity,e.g. a disorder of reduced motility of the gastrointestinal tract, in amammal.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides novel benzoimidazolone-carboxamide-derivedcarbamate 5-HT₄ receptor agonists of formula (I), orpharmaceutically-acceptable salts or solvates or stereoisomers thereof.The following substituents and values are intended to providerepresentative examples of various aspects of this invention. Theserepresentative values are intended to further define such aspects andare not intended to exclude other values or limit the scope of theinvention.

In specific aspects of the invention, R¹ is halo or C₁₋₃alkyl; or R¹ isfluoro, chloro, bromo, or methyl.

In a specific aspect, R² is hydrogen.

In another specific aspect, R² is C₁₋₃alkyl, wherein C₁₋₃alkyl isoptionally substituted with hydroxy.

In yet another specific aspect, R² is hydrogen or C₁₋₃alkyl.

In other specific aspects of the invention, R² is methyl, ethyl, propyl,or isopropyl; R² is ethyl or isopropyl; or R² is isopropyl.

In specific aspects, R³ is C₁₋₃alkyl; R³ is methyl or ethyl; or R³ ismethyl.

In a specific aspect, R⁴ is —(CH₂)₁₋₃C(O)NR^(a)R^(b).

In another specific aspect, R⁴ is

In yet another specific aspect, R⁴ is —(CH₂)₁₋₃C(O)NR^(a)R^(b) or

In a specific aspect of the invention, R³ and R⁴ together with thenitrogen atom to which they are attached form a moiety selected from amoiety of formula (a), a moiety of formula (b), and a moiety of formula(c).

In a specific aspect, R³ and R⁴ together with the nitrogen atom to whichthey are attached form a moiety of formula (a). In another specificaspect, R³ and R⁴ together with the nitrogen atom to which they areattached form a moiety of formula (a) wherein R⁵ is —OC(O)NR^(a)R^(b) or—C(O)NR^(a)R^(b).

In a specific aspect, R³ and R⁴ together with the nitrogen atom to whichthey are attached form a moiety of formula (b). In other specificaspects, R³ and R⁴ together with the nitrogen atom to which they areattached form a moiety of formula (b), wherein R⁶ is —C(O)R^(f),—(CH₂)₂OR^(g), or —S(O)₂NR^(a)R^(b); or R⁶ is —C(O)R^(f), and e is 1.

In still another aspect of the invention, R³ and R⁴ together with thenitrogen atom to which they are attached form a moiety of formula (c).

In specific aspects, R^(a), R^(b), R^(e), R^(d), and R^(g) areindependently hydrogen, methyl, or ethyl; or R^(a), R^(b), R^(c), R^(d),and R^(g) are independently hydrogen or methyl.

In specific aspects, R^(e) is methyl or ethyl; or R^(e) is methyl.

In specific aspects, R^(f) is C₁₋₃alkyl, tetrahydrofuranyl, or—NR^(a)R^(b); or R^(f) is methyl, tetrahydrofuranyl, or —NR^(a)R^(b).

In another specific aspect, R^(f) is tetrahydrofuranyl.

In other specific aspects, R^(f) is C₁₋₃alkyl, or R^(f) is methyl.

In yet another specific aspect, R^(f) is —NR^(a)R^(b), wherein R^(a) andR^(b) are as defined herein.

In specific aspects, a is 0 or 1; or a is 0 or 2. In another specificaspect, a is 0.

In specific aspects, b is 0, 1, or 2; or b is 1 or 2. In anotherspecific aspect, b is 1.

In a specific aspect, c is 1 or 2. In another specific aspect, c is 1.

In a specific aspect, d is 1.

In a specific aspect, e is 1.

In one aspect, the invention provides a compound of formula (I) whereinc is 1 or 2; d is 1; and e is 1.

In another aspect, the invention provides a compound of formula (I)wherein:

R³ is C₁₋₃alkyl; and

R⁴ is —(CH₂)₁₋₃C(O)NR^(a)R^(b) or

or R³ and R⁴ together with the nitrogen atom to which they are attachedform a moiety selected from a moiety of formula (a), a moiety of formula(b), and a moiety of formula (c);

wherein:

R⁵ is —OC(O)NR^(a)R^(b) or —C(O)NR^(a)R^(b); and

R⁶ is —C(O)R^(f), —(CH₂)₂OR^(g), or —S(O)₂NR^(a)R^(b).

In still another aspect, the invention provides a compound of formula(I) wherein R³ and R⁴ together with the nitrogen atom to which they areattached form a moiety selected from a moiety of formula (a), a moietyof formula (b), and a moiety of formula (c); wherein:

R⁵ is —OC(O)NR^(a)R^(b) or —C(O)NR^(a)R^(b);

R⁶ is —C(O)R^(f), —(CH₂)₂OR^(g), or —S(O)₂NR^(a)R^(b);

R^(a), R^(b), and R^(g) are independently hydrogen or methyl;

R^(f) is methyl, tetrahydrofuranyl, or —NR^(a)R^(b);

c is 1 or 2; d is 1; and e is 1.

In yet another aspect, the invention provides a compound of formula (I)wherein:

R² is ethyl or isopropyl

R³ is C₁₋₃alkyl; and

R⁴ is —(CH₂)₁₋₃C(O)NR^(a)R^(b) or

or R³ and R⁴ together with the nitrogen atom to which they are attachedform a moiety selected from a moiety of formula (a), a moiety of formula(b), and a moiety of formula (c); wherein

R⁵ is —OC(O)NR^(a)R^(b) or —C(O)NR^(a)R^(b);

R⁶ is —C(O)R^(f), —(CH₂)₂OR^(g), or —S(O)₂NR^(a)R^(b);

R^(a), R^(b), and R^(g) are independently hydrogen or methyl;

R^(f) is methyl, tetrahydrofuranyl, or —NR^(a)R^(b);

a is 0; c is 1 or 2; d is 1; and e is 1.

The chemical naming conventions used herein are illustrated for thecompound of Example 1

which is designated4-(tetrahydrofuran-2-carbonyl)piperazine-1-carboxylic acid3-{(1S,3R,5R)-3-[(3-isopropyl-2-oxo-2,3-dihydrobenzoimidazole-1-carbonyl)amino]-8-aza-bicyclo[3.2.1]oct-8-yl}propylester, according to the AutoNom software, provided by MDL InformationSystems, GmbH (Frankfurt, Germany). The designation (1S,3R,5R) describesthe relative orientation of the bonds associated with the bicyclic ringsystem that are depicted as solid and dashed wedges. In all of thecompounds of the invention depicted above, thebenzoimidazolone-carboxamide is endo to the azabicyclooctane group.

Particular mention may be made of the following compounds:

-   4-(tetrahydrofuran-2-carbonyl)piperazine-1-carboxylic acid    3-{(1S,3R,5R)-3-[(3-isopropyl-2-oxo-2,3-dihydrobenzoimidazole-1-carbonyl)amino]-8-azabicyclo-[3.2.1]oct-8-yl}propyl    ester;-   4-(2-hydroxyethyl)piperazine-1-carboxylic acid    3-{(1S,3R,5R)-3-[(3-isopropyl-2-oxo-2,3-dihydrobenzoimidazole-1-carbonyl)amino]-8-azabicyclo-[3.2.1]oct-8-yl}propyl    ester;-   4-acetyl-piperazine-1-carboxylic acid    3-{(1S,3R,5R)-3-[(3-isopropyl-2-oxo-2,3-dihydro-benzoimidazole-1-carbonyl)amino]-8-azabicyclo[3.2.1]oct-8-yl}propyl    ester;-   4-dimethylcarbamoyloxypiperidine-1-carboxylic acid    3-{(1S,3R,5R)-3-[(3-isopropyl-2-oxo-2,3-dihydrobenzoimidazole-1-carbonyl)amino]-8-aza-bicyclo[3.2.1]oct-8-yl}propyl    ester;-   3-carbamoylpiperidine-1-carboxylic acid    3-{(1S,3R,5R)-3-[(3-isopropyl-2-oxo-2,3-dihydro-benzoimidazole-1-carbonyl)amino]-8-azabicyclo[3.2.1]oct-8-yl}propyl    ester;-   1,1-dioxo-1λ⁶-thiomorpholine-4-carboxylic acid    3-{(1S,3R,5R)-3-[(3-isopropyl-2-oxo-2,3-dihydrobenzoimidazole-1-carbonyl)amino]-8-azabicyclo[3.2.1]oct-8-yl}propyl    ester;-   (1,1-dioxotetrahydro-1λ⁶-thiophen-3-yl)methylcarbamic acid    3-{(1S,3R,5R)-3-[(3-isopropyl-2-oxo-2,3-dihydrobenzoimidazole-1-carbonyl)amino]-8-azabicyclo-[3.2.1]oct-8-yl}propyl    ester;-   (R)-2-carbamoylpyrrolidine-1-carboxylic acid    3-{(1S,3R,5R)-3-[(3-isopropyl-2-oxo-2,3-dihydrobenzoimidazole-1-carbonyl)amino]-8-azabicyclo[3.2.1]oct-8-yl}propyl    ester;-   4-acetyl-[1,4]diazepane-1-carboxylic acid    3-{(1S,3R,5R)-3-[(3-isopropyl-2-oxo-2,3-dihydro-benzoimidazole-1-carbonyl)amino]-8-azabicyclo[3.2.1]oct-8-yl}propyl    ester;-   dimethylcarbamoylmethyl-methylcarbamic acid    3-{(1S,3R,5R)-3-[(3-isopropyl-2-oxo-2,3-dihydrobenzoimidazole-1-carbonyl)amino]-8-azabicyclo[3.2.1]oct-8-yl}propyl    ester;-   4-dimethylcarbamoylpiperazine-1-carboxylic acid    3-{(1S,3R,5R)-3-[(3-isopropyl-2-oxo-2,3-dihydrobenzoimidazole-1-carbonyl)amino]-8-azabicyclo[3.2.1]oct-8-yl}propyl    ester; and-   4-dimethylsulfamoylpiperazine-1-carboxylic acid    3-{(1S,3R,5R)-3-[(3-isopropyl-2-oxo-2,3-dihydrobenzoimidazole-1-carbonyl)amino]-8-azabicyclo[3.2.1]oct-8-yl}propyl    ester.

As exemplified by particular compounds listed above, the compounds ofthe invention may contain one or more chiral centers. Accordingly, theinvention includes racemic mixtures, pure stereoisomers, andstereoisomer-enriched mixtures of such isomers, unless otherwiseindicated. When a particular stereoisomer is shown, it will beunderstood by those skilled in the art, that minor amounts of otherstereoisomers may be present in the compositions of the invention unlessotherwise indicated, provided that any utility of the composition as awhole is not eliminated by the presence of such other isomers.

DEFINITIONS

When describing the compounds, compositions and methods of theinvention, the following terms have the following meanings, unlessotherwise indicated.

The term “alkyl” means a monovalent saturated hydrocarbon group whichmay be linear or branched or combinations thereof. Representative alkylgroups include, by way of example, methyl, ethyl, n-propyl (n-Pr),isopropyl (i-Pr), n-butyl (n-Bu), sec-butyl, isobutyl, tert-butyl, andthe like.

The term “halo” means fluoro, chloro, bromo or iodo.

The term “compound” means a compound that was synthetically prepared orproduced in any other way, such as by metabolism.

The term “therapeutically effective amount” means an amount sufficientto effect treatment when administered to a patient in need of treatment.

The term “treatment” as used herein means the treatment of a disease,disorder, or medical condition in a patient, such as a mammal(particularly a human) which includes:

-   -   (a) preventing the disease, disorder, or medical condition from        occurring, i.e., prophylactic treatment of a patient;    -   (b) ameliorating the disease, disorder, or medical condition,        i.e., eliminating or causing regression of the disease,        disorder, or medical condition in a patient;    -   (c) suppressing the disease, disorder, or medical condition,        i.e., slowing or arresting the development of the disease,        disorder, or medical condition in a patient; or    -   (d) alleviating the symptoms of the disease, disorder, or        medical condition in a patient.

The term “pharmaceutically-acceptable salt” means a salt prepared froman acid or base which is acceptable for administration to a patient,such as a mammal. Such salts can be derived frompharmaceutically-acceptable inorganic or organic acids and frompharmaceutically-acceptable bases. Typically,pharmaceutically-acceptable salts of compounds of the present inventionare prepared from acids.

Salts derived from pharmaceutically-acceptable acids include, but arenot limited to, acetic, adipic, benzenesulfonic, benzoic,camphorsulfonic, citric, ethanesulfonic, fumaric, gluconic, glutamic,hydrobromic, hydrochloric, lactic, maleic, malic, mandelic,methanesulfonic, mucic, nitric, pantothenic, phosphoric, succinic,sulfuric, tartaric, p-toluenesulfonic, xinafoic (1-hydroxy-2-naphthoicacid), naphthalene-1,5-disulfonic acid and the like.

The term “solvate” means a complex or aggregate formed by one or moremolecules of a solute, i.e. a compound of the invention or apharmaceutically-acceptable salt thereof, and one or more molecules of asolvent. Such solvates are typically crystalline solids having asubstantially fixed molar ratio of solute and solvent. Representativesolvents include by way of example, water, methanol, ethanol,isopropanol, acetic acid, and the like. When the solvent is water, thesolvate formed is a hydrate.

It will be appreciated that the term “or a pharmaceutically-acceptablesalt or solvate of stereoisomer thereof” is intended to include allpermutations of salts, solvates and stereoisomers, such as a solvate ofa pharmaceutically-acceptable salt of a stereoisomer of a compound offormula (I).

The term “leaving group” means a functional group or atom which can bedisplaced by another functional group or atom in a substitutionreaction, such as a nucleophilic substitution reaction. By way ofexample, representative leaving groups include chloro, bromo and iodogroups; sulfonic ester groups, such as mesylate, tosylate, brosylate,nosylate and the like; acyloxy groups, such as acetoxy, trifluoroacetoxyand the like. The term “leaving group” further encompasses groups suchas —OC₆F₅, —CCl₃, para-OC₆H₄NO₂, and imidazolyl.

The term “protected derivative thereof” means a derivative of thespecified compound in which one or more functional groups of thecompound are protected from undesired reactions with a protecting orblocking group. Functional groups which may be protected include, by wayof example, carboxylic acid groups, amino groups, hydroxyl groups, thiolgroups, carbonyl groups and the like. Representative protecting groupsfor carboxylic acids include esters (such as a p-methoxybenzyl ester),amides and hydrazides; for amino groups, carbamates (such astert-butoxycarbonyl) and amides; for hydroxyl groups, ethers and esters;for thiol groups, thioethers and thioesters; for carbonyl groups,acetals and ketals; and the like. Such protecting groups are well-knownto those skilled in the art and are described, for example, in T. W.Greene and G. M. Wuts, Protecting Groups in Organic Synthesis, ThirdEdition, Wiley, New York, 1999, and references cited therein.

The term “amino-protecting group” means a protecting group suitable forpreventing undesired reactions at an amino nitrogen. Representativeamino-protecting groups include, but are not limited to, formyl; acylgroups, for example alkanoyl groups, such as acetyl; alkoxycarbonylgroups, such as tert-butoxycarbonyl (Boc); arylmethoxycarbonyl groups,such as benzyloxycarbonyl (Cbz) and 9-fluorenylmethoxycarbonyl (Fmoc);arylmethyl groups, such as benzyl (Bn), trityl (Tr), and1,1-di-(4′-methoxyphenyl)methyl; silyl groups, such as trimethylsilyl(TMS) and tert-butyldimethylsilyl (TBDMS); and the like.

General Synthetic Procedures

Compounds of the invention can be prepared from readily availablestarting materials using the following general methods and procedures.Although a particular aspect of the present invention is illustrated inthe schemes below, those skilled in the art will recognize that allaspects of the present invention can be prepared using the methodsdescribed herein or by using other methods, reagents and startingmaterials known to those skilled in the art. It will also be appreciatedthat where typical or preferred process conditions (i.e., reactiontemperatures, times, mole ratios of reactants, solvents, pressures,etc.) are given, other process conditions can also be used unlessotherwise stated. Optimum reaction conditions may vary with theparticular reactants or solvent used, but such conditions can bedetermined by one skilled in the art by routine optimization procedures.

Additionally, as will be apparent to those skilled in the art,conventional protecting groups may be necessary to prevent certainfunctional groups from undergoing undesired reactions. The choice of asuitable protecting group for a particular functional group, as well assuitable conditions for protection and deprotection, are well known inthe art. For example, numerous protecting groups, and their introductionand removal, are described in T. W. Greene and G. M. Wuts, ProtectingGroups in Organic Synthesis, Third Edition, Wiley, New York, 1999, andreferences cited therein.

The substituents and variables shown in the following schemes have thedefinitions provided herein unless otherwise indicated.

In one method of synthesis, compounds of formula (I) can be prepared asillustrated in Scheme A.

A benzoimidazolone-carboxamide tropane intermediate (III) is reactedwith a compound of formula (IV), wherein L¹ is a leaving group, toprovide a compound of formula (I). Typically, L¹ is an S_(N)2-favoringleaving group, such as chloro, iodo, or bromo. A compound of formula(IV) is contacted with between about 0.25 and about 1.5 equivalents ofthe benzoimidazolone-carboxamide tropane (III), in an inert diluent, inthe presence of a base, such as N,N-diisopropylethylamine (DIPEA), andof a catalyst, such as sodium iodide. Suitable inert diluents includedimethylformamide, acetonitrile, tetrahydrofuran,N-methyl-2-pyrrolidone, and the like. Suitable bases also include, forexample, triethylamine, 1,8-diazabicyclo-[5.4.0]undec-7-ene (DBU), andpotassium carbonate. Suitable catalysts also include, for example,potassium iodide, and tetrabutylammonium iodide. This reaction istypically conducted at a temperature of from about 40° C. to about 100°C. for between about 2 and about 24 hours or until the reaction issubstantially complete.

The product of formula (I) is isolated and purified by conventionalprocedures. For example, the product can be concentrated to drynessunder reduced pressure, taken up in an aqueous weak acid solution andpurified by high-performance liquid chromatography (HPLC).

It will be understood that in the process of Scheme A and in otherprocesses described herein using a compound of formula (II), a compoundof formula (I) can be supplied in the form of the free base or in a saltform, with appropriate adjustment of reaction conditions, as necessary,as known to those skilled in the art.

A compound of formula (IV) can be prepared as shown in Scheme B below.

In Scheme B, intermediate (a), an optionally substituted1,3-dihydrobenzo-imidazol-2-one, is reacted with intermediate (b),wherein W is a leaving group (such as halo, i.e., fluoro, chloro, orbromo), and A is a leaving group chosen such that it reacts underdifferent conditions than W (such as —OC₆F₅, —CCl₃, para-OC₆H₄NO₂, orimidazol-1-yl); or Wand A are each imidazol-1-yl; to provide a compoundof formula (V), which is reacted with intermediate (c), wherein P¹represents an amino protecting group, such as Boc, to provideintermediate (d). The protecting group P¹ is removed from intermediate(d) by standard procedures to provide a compound of formula (III).

While optimum reaction conditions may vary depending on the particularreactants or solvents used, such conditions can be readily determined byone skilled in the art by routine optimization procedures.

For example, in an exemplary process using 4-nitrophenyl chloroformateas intermediate (b), the benzoimidazolone intermediate (a) is dissolvedunder an inert atmosphere in an inert diluent, such as tetrahydrofuran,ether, DMF, or a combination thereof, in the presence of a strong base,such as sodium hydride, lithium diisopropylamine, and n-butyl lithium,and is contacted with between about 1 and about 1.3 equivalents of4-nitrophenyl chloroformate. The mixture is stirred at about 0° C. toabout 40° C. for between about 12 and about 24 hours or until thereaction is substantially complete to form an activated ester, acompound of formula (V). The compound of formula (V) is isolated andpurified, or it can be reacted, in situ, with a protected amino-tropane,intermediate (c), in the presence of an inert diluent, such astetrahydrofuran, at a temperature of from about 30° C. to about 90° C.for between about 10 and about 24 hours to provide protectedintermediate (d).

Using conventional methods, the amino-protecting group, P¹ is removedfrom intermediate (d), to provide a benzoimidazolone-carboxamide tropanecompound of formula (III). A variation of the above process of preparingintermediate (III) using 4-nitrophenyl chloroformate as intermediate (b)is described in Example 13 below.

Alternatively, a compound of formula (I) can be prepared as shown inScheme C below.

A benzoimidazolone-carboxamide intermediate (V) is reacted with atropane-alkylene-carboxamide compound of formula (VI) to provide acompound of formula (I). The compound of formula (V), the synthesis ofwhich is described in Scheme B, is either isolated and purified, orreacted in situ, with a compound of formula (VI), in the presence of aninert diluent, such as tetrahydrofuran, at a temperature range of fromabout 30° C. to about 90° C. for between about 10 and about 24 hours, oruntil the reaction is complete, to provide a compound of formula (I).

The benzoimidazolone intermediate (a) can be prepared as shown below inScheme D.

In Scheme D, an optionally substituted 2-fluoronitrobenzene is reactedwith a primary amine, intermediate (e), to provide intermediate (f),which is reduced to a diaminophenyl, intermediate (g). The diaminophenylis reacted with carbonyldiimidazole in the presence of an inert diluent,such as tetrahydrofuran, at a temperature of from about 20° C. to about40° C. for between about 12 and about 30 hours, to provide abenzoimidazolone intermediate (a).

A representative synthesis of a compound of intermediate (a) isdescribed below in Preparation 1. A substituted compound of intermediate(a) can also readily be prepared by procedures similar to thosedescribed in the literature. See, for example, The Journal of ChemicalResearch (1), 21-22 (2005); Heteroatom Chemistry, 5(5/6):437-40 (1994);and Ger. Offen., 3839743, 31 May 1990.

The protected aminotropane, intermediate (c) employed in the reactionsdescribed in this application is prepared from readily availablestarting materials. For example, when the amino-protecting group P¹ isBoc, the protected aminotropane can be prepared as shown below in SchemeE.

As described in detail in Preparation 2 below, to prepare theBoc-protected intermediate (c′), 2,5-dimethoxytetrahydrofuran iscontacted with between about 1 and 2 equivalents of benzyl amine and aslight excess, for example about 1.1 equivalents, of1,3-acetonedicarboxylic acid in an acidic aqueous solution in thepresence of a buffering agent, such as sodium hydrogen phosphate. Thereaction mixture is heated to between about 60° C. and about 100° C. toensure decarboxylation of any carboxylated intermediates in the product,8-benzyl-8-azabicyclo-[3.2.1]octan-3-one, commonly N-benzyltropanone.

The N-benzyltropanone intermediate is typically reacted with a slightexcess of di-tert-butyl dicarbonate (commonly (Boc)₂O), for example,about 1.1 equivalents, under a hydrogen atmosphere in the presence of atransition metal catalyst to provide3-oxo-8-azabicyclo[3.2.1]octane-8-carboxylic acid tert-butyl ester. Thereaction is typically conducted at ambient temperature for about 12 toabout 72 hours. Finally, 3-oxo-8-azabicyclo[3.2.1]octane-8-carboxylicacid tert-butyl ester is contacted with a large excess, for example atleast about 25 equivalents, of ammonium formate in an inert diluent,such as methanol, in the presence of a transition metal catalyst toprovide the product, intermediate (c), in the endo configuration withhigh stereospecificity, for example endo to exo ratio of >99:1. Thereaction is typically conducted at ambient temperature for about 12 toabout 72 hours or until the reaction is substantially complete. It isadvantageous to add the ammonium formate reagent in portions. Forexample, 3-oxo-8-azabicyclo[3.2.1]octane-8-carboxylic acid tert-butylester is contacted with an initial portion of ammonium formate of about15 to about 25 equivalents. After an interval of about 12 to about 36hours, an additional portion of about 5 to about 10 equivalents ofammonium formate is added. The subsequent addition can be repeated aftera similar interval. The product, intermediate (c), can be purified byconventional procedures, such as alkaline extraction.

A compound of formula (IV) can be easily prepared by standard proceduresfrom common starting materials, as described below in Scheme F.

In Scheme F, intermediate (h), wherein L¹ and L² are leaving groups, isreacted with a secondary amine (i) to provide a compound of formula(IV). The secondary amine (i) is dissolved in an inert diluent, such asdichloromethane, and intermediate (h) is added, in the presence of abase, such as N,N-diisopropylethylamine, at a temperature ranging fromabout 0° C. to about 40° C., for about 30 minutes to about 4 hours. Theproduct, a compound of formula (IV), can be purified by conventionalprocedures, such as by HPLC.

Typically, L¹ and L² are halo leaving groups, such as chloro, iodo,bromo; mesylate can also be used as leaving group L¹. Suitable bases caninclude, for example, triethyl amine, DBU, and potassium carbonate.Suitable inert diluents can include acetonitrile, tetrahydrofuran, andN,N-dimethylformamide.

Intermediate compounds of formula (h) and (i) are available commerciallyor can be synthesized from readily available starting materials. Thesynthesis of many secondary amines, i.e., intermediate compounds offormula (I) that can be used in Scheme F, are described in the Examplessection herein.

A compound of formula (VI) can be prepared as shown in Scheme G.

In Scheme G, intermediate (O), wherein P² is an amino-protecting group,is reacted with a compound of formula (IV) to provide protectedintermediate (k), which is then deprotected to provide a compound offormula (VI). The reaction of Scheme G is typically conducted under theamine coupling conditions described above for the reaction of Scheme A.

A compound of formula (j) can be prepared by protecting the aminonitrogen of the protected aminotropane intermediate (c), with anamino-protecting group P² and then removing P¹ from the nitrogen of theazabicyclooctane group. Protecting groups P¹ and P² are chosen such thatthey are removed under different conditions. For example, when P¹ ischosen as Boc, then Cbz can be used as P². The protecting group Boc istypically removed by treatment with an acid, such as trifluoroaceticacid, providing the acid salt of the intermediate. The acid salt of theintermediate can be converted to the free base, if desired, byconventional treatment with base. The protecting group Cbz isconveniently removed by hydrogenolysis over a suitable metal catalystsuch as palladium on carbon. Further details regarding specific reactionconditions and other procedures for preparing representative compoundsof the invention or intermediates thereto are described in the examplesbelow.

Accordingly, in a method aspect, the invention provides a process forpreparing a compound of formula (I) or a salt or solvate or stereoisomerthereof, wherein R¹, R², R³, R⁴, a, and b are as defined herein, theprocess comprising:

(a) reacting a compound of formula (III):

-   -   with a compound of formula (IV):    -   wherein L¹ is a leaving group; or

(b) reacting a compound of formula (V):

-   -   wherein A is a leaving group;    -   with a compound of formula (VI):

to provide a compound of formula (I), or a salt or solvate orstereoisomer thereof.

In additional embodiments, this invention is directed to the otherprocesses described herein; and to the products prepared by any of theprocesses described herein.

Pharmaceutical Compositions

The benzoimidazolone-carboxamide-derived carbamate compounds of theinvention are typically administered to a patient in the form of apharmaceutical composition. Such pharmaceutical compositions may beadministered to the patient by any acceptable route of administrationincluding, but not limited to, oral, rectal, vaginal, nasal, inhaled,topical (including transdermal) and parenteral modes of administration.

Accordingly, in one of its compositions aspects, the invention isdirected to a pharmaceutical composition comprising apharmaceutically-acceptable carrier or excipient and a therapeuticallyeffective amount of a compound of formula (I) or a pharmaceuticallyacceptable salt thereof. Optionally, such pharmaceutical compositionsmay contain other therapeutic and/or formulating agents if desired.

The pharmaceutical compositions of the invention typically contain atherapeutically effective amount of a compound of the present inventionor a pharmaceutically-acceptable salt thereof. Typically, suchpharmaceutical compositions will contain from about 0.1 to about 95% byweight of the active agent; preferably, from about 5 to about 70% byweight; and more preferably from about 10 to about 60% by weight of theactive agent.

Any conventional carrier or excipient may be used in the pharmaceuticalcompositions of the invention. The choice of a particular carrier orexcipient, or combinations of carriers or excipients, will depend on themode of administration being used to treat a particular patient or typeof medical condition or disease state. In this regard, the preparationof a suitable pharmaceutical composition for a particular mode ofadministration is well within the scope of those skilled in thepharmaceutical arts. Additionally, the ingredients for such compositionsare commercially available from, for example, Sigma, P.O. Box 14508, St.Louis, Mo. 63178. By way of further illustration, conventionalformulation techniques are described in Remington: The Science andPractice of Pharmacy, 20^(th) Edition, Lippincott Williams & White,Baltimore, Md. (2000); and H. C. Ansel et al., Pharmaceutical DosageForms and Drug Delivery Systems, 7th Edition, Lippincott Williams &White, Baltimore, Md. (1999).

Representative examples of materials which can serve as pharmaceuticallyacceptable carriers include, but are not limited to, the following: (1)sugars, such as lactose, glucose and sucrose; (2) starches, such as cornstarch and potato starch; (3) cellulose, such as microcrystallinecellulose, and its derivatives, such as sodium carboxymethyl cellulose,ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5)malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter andsuppository waxes; (9) oils, such as peanut oil, cottonseed oil,safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10)glycols, such as propylene glycol; (11) polyols, such as glycerin,sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyloleate and ethyl laurate; (13) agar; (14) buffering agents, such asmagnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16)pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19)ethyl alcohol; (20) phosphate buffer solutions; and (21) other non-toxiccompatible substances employed in pharmaceutical compositions.

The pharmaceutical compositions of the invention are typically preparedby thoroughly and intimately mixing or blending a compound of theinvention with a pharmaceutically-acceptable carrier and one or moreoptional ingredients. If necessary or desired, the resulting uniformlyblended mixture can then be shaped or loaded into tablets, capsules,pills and the like using conventional procedures and equipment.

The pharmaceutical compositions of the invention are preferably packagedin a unit dosage form. The term “unit dosage form” means a physicallydiscrete unit suitable for dosing a patient, i.e., each unit containinga predetermined quantity of active agent calculated to produce thedesired therapeutic effect either alone or in combination with one ormore additional units. For example, such unit dosage forms may becapsules, tablets, pills, and the like.

In a preferred embodiment, the pharmaceutical compositions of theinvention are suitable for oral administration. Suitable pharmaceuticalcompositions for oral administration may be in the form of capsules,tablets, pills, lozenges, cachets, dragees, powders, granules; or as asolution or a suspension in an aqueous or non-aqueous liquid; or as anoil-in-water or water-in-oil liquid emulsion; or as an elixir or syrup;and the like; each containing a predetermined amount of a compound ofthe present invention as an active ingredient.

When intended for oral administration in a solid dosage form (i.e., ascapsules, tablets, pills and the like), the pharmaceutical compositionsof the invention will typically comprise a compound of the presentinvention as the active ingredient and one or morepharmaceutically-acceptable carriers, such as sodium citrate ordicalcium phosphate. Optionally or alternatively, such solid dosageforms may also comprise: (1) fillers or extenders, such as starches,microcrystalline cellulose, lactose, sucrose, glucose, mannitol, and/orsilicic acid; (2) binders, such as carboxymethylcellulose, alginates,gelatin, polyvinyl pyrrolidone, sucrose and/or acacia; (3) humectants,such as glycerol; (4) disintegrating agents, such as agar-agar, calciumcarbonate, potato or tapioca starch, alginic acid, certain silicates,and/or sodium carbonate; (5) solution retarding agents, such asparaffin; (6) absorption accelerators, such as quaternary ammoniumcompounds; (7) wetting agents, such as cetyl alcohol and/or glycerolmonostearate; (8) absorbents, such as kaolin and/or bentonite clay; (9)lubricants, such as talc, calcium stearate, magnesium stearate, solidpolyethylene glycols, sodium lauryl sulfate, and/or mixtures thereof;(10) coloring agents; and (11) buffering agents.

Release agents, wetting agents, coating agents, sweetening, flavoringand perfuming agents, preservatives and antioxidants can also be presentin the pharmaceutical compositions of the invention. Examples ofpharmaceutically-acceptable antioxidants include: (1) water-solubleantioxidants, such as ascorbic acid, cysteine hydrochloride, sodiumbisulfate, sodium metabisulfate sodium sulfite and the like; (2)oil-soluble antioxidants, such as ascorbyl palmitate, butylatedhydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propylgallate, alpha-tocopherol, and the like; and (3) metal-chelating agents,such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol,tartaric acid, phosphoric acid, and the like. Coating agents fortablets, capsules, pills and like, include those used for entericcoatings, such as cellulose acetate phthalate (CAP), polyvinyl acetatephthalate (PVAP), hydroxypropyl methylcellulose phthalate, methacrylicacid-methacrylic acid ester copolymers, cellulose acetate trimellitate(CAT), carboxymethyl ethyl cellulose (CMEC), hydroxypropyl methylcellulose acetate succinate (HPMCAS), and the like.

If desired, the pharmaceutical compositions of the present invention mayalso be formulated to provide slow or controlled release of the activeingredient using, by way of example, hydroxypropyl methyl cellulose invarying proportions; or other polymer matrices, liposomes and/ormicrospheres.

In addition, the pharmaceutical compositions of the present inventionmay optionally contain opacifying agents and may be formulated so thatthey release the active ingredient only, or preferentially, in a certainportion of the gastrointestinal tract, optionally, in a delayed manner.Examples of embedding compositions which can be used include polymericsubstances and waxes. The active ingredient can also be inmicro-encapsulated form, if appropriate, with one or more of theabove-described excipients.

Suitable liquid dosage forms for oral administration include, by way ofillustration, pharmaceutically-acceptable emulsions, microemulsions,solutions, suspensions, syrups and elixirs. Such liquid dosage formstypically comprise the active ingredient and an inert diluent, such as,for example, water or other solvents, solubilizing agents andemulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate,ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol,1,3-butylene glycol, oils (such as cottonseed, groundnut, corn, germ,olive, castor and sesame oils), glycerol, tetrahydrofuryl alcohol,polyethylene glycols and fatty acid esters of sorbitan, and mixturesthereof. Suspensions, in addition to the active ingredient, may containsuspending agents such as, for example, ethoxylated isostearyl alcohols,polyoxyethylene sorbitol and sorbitan esters, microcrystallinecellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth,and mixtures thereof.

Alternatively, the pharmaceutical compositions of the invention areformulated for administration by inhalation. Suitable pharmaceuticalcompositions for administration by inhalation will typically be in theform of an aerosol or a powder. Such compositions are generallyadministered using well-known delivery devices, such as a metered-doseinhaler, a dry powder inhaler, a nebulizer or a similar delivery device.

When administered by inhalation using a pressurized container, thepharmaceutical compositions of the invention will typically comprise theactive ingredient and a suitable propellant, such asdichlorodifluoromethane, trichlorofluoromethane,dichlorotetrafluoroethane, carbon dioxide or other suitable gas.

Additionally, the pharmaceutical composition may be in the form of acapsule or cartridge (made, for example, from gelatin) comprising acompound of the invention and a powder suitable for use in a powderinhaler. Suitable powder bases include, by way of example, lactose orstarch.

The compounds of the invention can also be administered transdermallyusing known transdermal delivery systems and excipients. For example, acompound of the invention can be admixed with permeation enhancers, suchas propylene glycol, polyethylene glycol monolaurate,azacycloalkan-2-ones and the like, and incorporated into a patch orsimilar delivery system. Additional excipients including gelling agents,emulsifiers and buffers, may be used in such transdermal compositions ifdesired.

The following formulations illustrate representative pharmaceuticalcompositions of the present invention:

FORMULATION EXAMPLE A

Hard gelatin capsules for oral administration are prepared as follows:Ingredients Amount Compound of the invention 50 mg Lactose (spray-dried)200 mg Magnesium stearate 10 mg

-   -   Representative Procedure: The ingredients are thoroughly blended        and then loaded into a hard gelatin capsule (260 mg of        composition per capsule).

FORMULATION EXAMPLE B

Hard gelatin capsules for oral administration are prepared as follows:Ingredients Amount Compound of the invention 20 mg Starch 89 mgMicrocrystalline cellulose 89 mg Magnesium stearate 2 mg

-   -   Representative Procedure: The ingredients are thoroughly blended        and then passed through a No. 45 mesh U.S. sieve and loaded into        a hard gelatin capsule (200 mg of composition per capsule).

FORMULATION EXAMPLE C

Capsules for oral administration are prepared as follows: IngredientsAmount Compound of the invention 10 mg Polyoxyethylene sorbitanmonooleate 50 mg Starch powder 250 mg

-   -   Representative Procedure: The ingredients are thoroughly blended        and then loaded into a gelatin capsule (310 mg of composition        per capsule).

FORMULATION EXAMPLE D

Tablets for oral administration are prepared as follows: IngredientsAmount Compound of the invention 5 mg Starch 50 mg Microcrystallinecellulose 35 mg Polyvinylpyrrolidone (10 wt. % in water) 4 mg Sodiumcarboxymethyl starch 4.5 mg Magnesium stearate 0.5 mg Talc 1 mg

Representative Procedure: The active ingredient, starch and celluloseare passed through a No. 45 mesh U.S. sieve and mixed thoroughly. Thesolution of polyvinylpyrrolidone is mixed with the resulting powders,and this mixture is then passed through a No. 14 mesh U.S. sieve. Thegranules so produced are dried at 50-60° C. and passed through a No. 18mesh U.S. sieve. The sodium carboxymethyl starch, magnesium stearate andtalc (previously passed through a No. 60 mesh U.S. sieve) are then addedto the granules. After mixing, the mixture is compressed on a tabletmachine to afford a tablet weighing 100 mg.

FORMULATION EXAMPLE E

Tablets for oral administration are prepared as follows: IngredientsAmount Compound of the invention 25 mg Microcrystalline cellulose 400 mgSilicon dioxide fumed 10 mg Stearic acid 5 mg

-   -   Representative Procedure: The ingredients are thoroughly blended        and then compressed to form tablets (440 mg of composition per        tablet).

FORMULATION EXAMPLE F

Single-scored tablets for oral administration are prepared as follows:Ingredients Amount Compound of the invention 15 mg Cornstarch 50 mgCroscarmellose sodium 25 mg Lactose 120 mg Magnesium stearate 5 mg

-   -   Representative Procedure: The ingredients are thoroughly blended        and compressed to form a single-scored tablet (215 mg of        compositions per tablet).

FORMULATION EXAMPLE G

A suspension for oral administration is prepared as follows: IngredientsAmount Compound of the invention 0.1 g Fumaric acid 0.5 g Sodiumchloride 2.0 g Methyl paraben 0.15 g Propyl paraben 0.05 g Granulatedsugar 25.5 g Sorbitol (70% solution) 12.85 g Veegum k (Vanderbilt Co.)1.0 g Flavoring 0.035 mL Colorings 0.5 mg Distilled water q.s. to 100 mL

-   -   Representative Procedure: The ingredients are mixed to form a        suspension containing 10 mg of active ingredient per 10 mL of        suspension.

FORMULATION EXAMPLE H

A dry powder for administration by inhalation is prepared as follows:Ingredients Amount Compound of the invention 1.0 mg Lactose 25 mg

-   -   Representative Procedure: The active ingredient is micronized        and then blended with lactose. This blended mixture is then        loaded into a gelatin inhalation cartridge. The contents of the        cartridge are administered using a powder inhaler.

FORMULATION EXAMPLE I

A dry powder for administration by inhalation in a metered dose inhaleris prepared as follows:

-   -   Representative Procedure: A suspension containing 5 wt. % of a        compound of the invention and 0.1 wt. % lecithin is prepared by        dispersing 10 g of active compound as micronized particles with        mean size less than 10 μm in a solution formed from 0.2 g of        lecithin dissolved in 200 mL of demineralized water. The        suspension is spray dried and the resulting material is        micronized to particles having a mean diameter less than 1.5 μm.        The particles are loaded into cartridges with pressurized        1,1,1,2-tetrafluoroethane.

FORMULATION EXAMPLE J

An injectable formulation is prepared as follows: Ingredients AmountCompound of the invention 0.2 g Sodium acetate buffer solution (0.4 M)40 mL HCl (0.5 N) or NaOH (0.5 N) q.s. to pH 4 Water (distilled,sterile) q.s. to 20 mL

-   -   Representative Procedure: The above ingredients are blended and        the pH is adjusted to 4±0.5 using 0.5 N HCl or 0.5 N NaOH.

FORMULATION EXAMPLE K

Capsules for oral administration are prepared as follows: IngredientsAmount Compound of the Invention 4.05 mg Microcrystalline cellulose(Avicel PH 103) 259.2 mg Magnesium stearate 0.75 mg

-   -   Representative Procedure: The ingredients are thoroughly blended        and then loaded into a gelatin capsule (Size #1, White, Opaque)        (264 mg of composition per capsule).

FORMULATION EXAMPLE L

Capsules for oral administration are prepared as follows: IngredientsAmount Compound of the Invention 8.2 mg Microcrystalline cellulose(Avicel PH 103) 139.05 mg Magnesium stearate 0.75 mg

-   -   Representative Procedure: The ingredients are thoroughly blended        and then loaded into a gelatin capsule (Size #1, White, Opaque)        (148 mg of composition per capsule).

It will be understood that any form of the compounds of the invention,(i.e. free base, pharmaceutical salt, or solvate) that is suitable forthe particular mode of administration, can be used in the pharmaceuticalcompositions discussed above.

Utility

The benzoimidazolone-carboxamide-derived carbamate compounds of theinvention are 5-HT₄ receptor agonists and therefore are expected to beuseful for treating medical conditions mediated by 5-HT₄ receptors orassociated with 5-HT₄ receptor activity, i.e. medical conditions whichare ameliorated by treatment with a 5-HT₄ receptor agonist. Such medicalconditions include, but are not limited to, irritable bowel syndrome(IBS), chronic constipation, functional dyspepsia, delayed gastricemptying, gastroesophageal reflux disease (GERD), gastroparesis,post-operative ileus, intestinal pseudo-obstruction, and drug-induceddelayed transit. In addition, it has been suggested that some 5-HT₄receptor agonist compounds may be used in the treatment of centralnervous system disorders including cognitive disorders, behavioraldisorders, mood disorders, and disorders of control of autonomicfunction.

In particular, the compounds of the invention increase motility of thegastrointestinal (GI) tract and thus are expected to be useful fortreating disorders of the GI tract caused by reduced motility inmammals, including humans. Such GI motility disorders include, by way ofillustration, chronic constipation, constipation-predominant irritablebowel syndrome (C-EBS), diabetic and idiopathic gastroparesis, andfunctional dyspepsia.

In one aspect, therefore, the invention provides a method of increasingmotility of the gastrointestinal tract in a mammal, the methodcomprising administering to the mammal a therapeutically effectiveamount of a pharmaceutical composition comprising apharmaceutically-acceptable carrier and a compound of the invention.

When used to treat disorders of reduced motility of the GI tract orother conditions mediated by 5-HT₄ receptors, the compounds of theinvention will typically be administered orally in a single daily doseor in multiple doses per day, although other forms of administration maybe used. The amount of active agent administered per dose or the totalamount administered per day will typically be determined by a physician,in the light of the relevant circumstances, including the condition tobe treated, the chosen route of administration, the actual compoundadministered and its relative activity, the age, weight, and response ofthe individual patient, the severity of the patient's symptoms, and thelike.

Suitable doses for treating disorders of reduced motility of the GItract or other disorders mediated by 5-HT₄ receptors will range fromabout 0.0007 to about 20 mg/kg/day of active agent, including from about0.0007 to about 1 mg/kg/day. For an average 70 kg human, this wouldamount to from about 0.05 to about 70 mg per day of active agent.

In one aspect of the invention, the compounds of the invention are usedto treat chronic constipation. When used to treat chronic constipation,the compounds of the invention will typically be administered orally ina single daily dose or in multiple doses per day. Preferably, the dosefor treating chronic constipation will range from about 0.05 to about 70mg per day.

In another aspect of the invention, the compounds of the invention areused to treat irritable bowel syndrome. When used to treatconstipation-predominant irritable bowel syndrome, the compounds of theinvention will typically be administered orally in a single daily doseor in multiple doses per day. Preferably, the dose for treatingconstipation-predominant irritable bowel syndrome will range from about0.05 to about 70 mg per day.

In another aspect of the invention, the compounds of the invention areused to treat diabetic gastroparesis. When used to treat diabeticgastroparesis, the compounds of the invention will typically beadministered orally in a single daily dose or in multiple doses per day.Preferably, the dose for treating diabetic gastroparesis will range fromabout 0.05 to about 70 mg per day.

In yet another aspect of the invention, the compounds of the inventionare used to treat functional dyspepsia. When used to treat functionaldyspepsia, the compounds of the invention will typically be administeredorally in a single daily dose or in multiple doses per day. Preferably,the dose for treating functional dyspepsia will range from about 0.05 toabout 70 mg per day.

The invention also provides a method of treating a mammal having adisease or condition associated with 5-HT₄ receptor activity, the methodcomprising administering to the mammal a therapeutically effectiveamount of a compound of the invention or of a pharmaceutical compositioncomprising a compound of the invention.

Since compounds of the invention are 5-HT₄ receptor agonists, suchcompounds are also useful as research tools for investigating orstudying biological systems or samples having 5-HT₄ receptors, or fordiscovering new 5-HT₄ receptor agonists. Moreover, since compounds ofthe invention exhibit binding selectivity for 5-HT₄ receptors ascompared with binding to receptors of other 5-HT subtypes, particularly5-HT₃ receptors, such compounds are particularly useful for studying theeffects of selective agonism of 5-HT₄ receptors in a biological systemor sample. Any suitable biological system or sample having 5-HT₄receptors may be employed in such studies which may be conducted eitherin vitro or in vivo. Representative biological systems or samplessuitable for such studies include, but are not limited to, cells,cellular extracts, plasma membranes, tissue samples, mammals (such asmice, rats, guinea pigs, rabbits, dogs, pigs, etc.) and the like.

In this aspect of the invention, a biological system or samplecomprising a 5-HT₄ receptor is contacted with a 5-HT₄ receptor-agonizingamount of a compound of the invention. The effects of agonizing the5-HT₄ receptor are then determined using conventional procedures andequipment, such as radioligand binding assays and functional assays.Such functional assays include ligand-mediated changes in intracellularcyclic adenosine monophosphate (cAMP), ligand-mediated changes inactivity of the enzyme adenylyl cyclase (which synthesizes cAMP),ligand-mediated changes in incorporation of analogs of guanosinetriphosphate (GTP), such as [³⁵S]GTPγS (guanosine5′-O-(γ-thio)triphosphate) or GTP-Eu, into isolated membranes viareceptor catalyzed exchange of GTP analogs for GDP analogs,ligand-mediated changes in free intracellular calcium ions (measured,for example, with a fluorescence-linked imaging plate reader or FLIPR®from Molecular Devices, Inc.), and measurement of mitogen activatedprotein kinase (MAPK) activation. A compound of the invention mayagonize or increase the activation of 5-HT₄ receptors in any of thefunctional assays listed above, or assays of a similar nature. A 5-HT₄receptor-agonizing amount of a compound of the invention will typicallyrange from about 1 nanomolar to about 500 nanomolar.

Additionally, the compounds of the invention can be used as researchtools for discovering new 5-HT₄ receptor agonists. In this embodiment,5-HT₄ receptor binding or functional data for a test compound or a groupof test compounds is compared to the 5-HT₄ receptor binding orfunctional data for a compound of the invention to identify testcompounds that have superior binding or functional activity, if any.This aspect of the invention includes, as separate embodiments, both thegeneration of comparison data (using the appropriate assays) and theanalysis of the test data to identify test compounds of interest.

Among other properties, compounds of the invention have been found to bepotent agonists of the 5-HT₄ receptor and to exhibit substantialselectivity for the 5-HT₄ receptor subtype over the 5-HT₃ receptorsubtype in radioligand binding assays. Further, representative compoundsof the invention have demonstrated superior pharmacokinetic propertiesin a rat model. Compounds of the invention are thus expected todemonstrate good bioavailability upon oral administration. In addition,representative compounds of the invention tested in an in vitrovoltage-clamp model using isolated whole cells expressing the hERGcardiac potassium channel, have been shown not to exhibit anunacceptable level of inhibition of the potassium ion current. Thevoltage-clamp assay is an accepted pre-clinical method of assessing thepotential for pharmaceutical agents to change the pattern of cardiacrepolarization, specifically to cause, so-called QT prolongation, whichhas been associated with cardiac arrhythmia. (Cavero et al., Opinion onPharmacotherapy, 2000, 1, 947-73, Fermini et al., Nature Reviews DrugDiscovery, 2003, 2, 439-447) Accordingly, pharmaceutical compositionscomprising compounds of the invention are expected to have an acceptablecardiac profile.

These properties, as well as the utility of the compounds of theinvention, can be demonstrated using various in vitro and in vivo assayswell-known to those skilled in the art. Representative assays aredescribed in further detail in the following examples.

EXAMPLES

The following synthetic and biological examples are offered toillustrate the invention, and are not to be construed in any way aslimiting the scope of the invention. In the examples below, thefollowing abbreviations have the following meanings unless otherwiseindicated. Abbreviations not defined below have their generally acceptedmeanings.

-   -   Boc=tert-butoxycarbonyl    -   (Boc)₂O=di-tert-butyl dicarbonate    -   DCM=dichloromethane    -   DMF=N,N-dimethylformamide    -   DMSO=dimethyl sulfoxide    -   EtOAc=ethyl acetate    -   mCPBA=m-chloroperbenzoic acid    -   MeCN=acetonitrile    -   MTBE=tert-butyl methyl ether    -   PyBop=benzotriazol-1-yloxytripyrrolidino-phosphonium        hexafluorophosphate    -   R_(f)=retention factor    -   RT=room temperature    -   TFA=trifluoroacetic acid    -   THF=tetrahydrofuran

Reagents (including secondary amines) and solvents were purchased fromcommercial suppliers (Aldrich, Fluka, Sigma, etc.), and used withoutfurther purification. Reactions were run under nitrogen atmosphere,unless noted otherwise. Progress of reaction mixtures was monitored bythin layer chromatography (TLC), analytical high performance liquidchromatography (anal. HPLC), and mass spectrometry, the details of whichare given below and separately in specific examples of reactions.Reaction mixtures were worked up as described specifically in eachreaction; commonly they were purified by extraction and otherpurification methods such as temperature-, and solvent-dependentcrystallization, and precipitation. In addition, reaction mixtures wereroutinely purified by preparative HPLC: a general protocol is describedbelow. Characterization of reaction products was routinely carried outby mass and ¹H-NMR spectrometry. For NMR measurement, samples weredissolved in deuterated solvent (CD₃OD, CDCl₃, or DMSO-d₆), and ¹H-NMRspectra were acquired with a Varian Gemini 2000 instrument (300 MHz)under standard observation conditions. Unless otherwise indicated, massspectrometric identification of compounds was performed by anelectrospray ionization method (ESMS) with an Applied Biosystems (FosterCity, Calif.) model API 150 EX instrument or an Agilent (Palo Alto,Calif.) model 1100 LC/MSD instrument.

A general protocol for analytical HPLC: Each of crude compounds wasdissolved in 50% MeCN/H₂O (with 0.1% TFA) at 0.5-1.0 mg/mLconcentration, and was analyzed by using anal. HPLC: 1) reverse-phasedanal. Column: Zorbax Bonus-RP (3.5 μm of particle size, 2.1×50 mm); 2)flow rate: 0.5 mL/min; 3) 5% MeCN/H₂O containing 0.1% TFA (isocratic;0-0.5 min); 5% MeCN/H₂O containing 0.1% TFA to 75% MeCN/H₂O containing0.1% TFA (linear gradient; 0.5-4 min); 4) detection: 214, 254, and 280nm. Other conditions used are indicated whenever necessary.

A general protocol for preparative HPLC purification: Crude compoundswere dissolved in 50% acetic acid in water at 50-100 mg/mLconcentration, filtered, and fractionated using preparative HPLC: 1)column; YMC Pack-Pro C18 (50a×20 mm; ID=5 μm); 2) linear gradient: 10%A/90% B to 50% A150% B over 30 min; 3) flow rate: 40 mL/min; 4)detection: 214 nm.

Preparation of Secondary Amines

Preparation of Various Secondary Amines Used as Intermediates in theSynthesis of a compound of formula (I) are described below.

The N-sulfonyl derivatives of piperazine were prepared from N-Bocpiperazine by reacting with respective sulfonyl chloride (iPr₂NEt,CH₂Cl₂, 0° C.), and deprotecting the N-Boc group (CF₃CO₂H, CH₂Cl₂).1-Methanesulfonylpiperazine: ¹H-NMR (CDCl₃; neutral): δ (ppm) 3.1 (t,4H), 2.9 (t, 4H), 2.7 (s, 3H). Methanesulfonylpiperazine was alsoprepared by reacting methanesulfonyl chloride with excess piperazine (>2equivalents) in water.

The N-derivatives of piperazine such as1-(dimethylaminocarbonyl)piperazine, and1-(dimethylaminosulfonyl)piperazine were prepared by reacting piperazinewith dimethylaminochloroformate, or dimethylaminosulfamoyl chloride,respectively.

The racemic or single chiral isomer forms of 3-acetylaminopyrrolidinewere prepared by treating N¹-Boc-3-aminopyrrolidine (racemate, 3R, or3S) with acetyl chloride (iPr₂NEt, CH₂Cl₂, 0° C.), and deprotecting theN-Boc group (CF₃CO₂H, CH₂Cl₂). 3-(Acetamido)pyrrolidine: ¹H-NMR(DMSO-d₆; TFA salt): δ (ppm) 4.2 (quin, 1H), 3.3-3.1 (m, 3H), 2.9 (m,1H), 2.0 (m, 1H), 1.8 (br s, 4H).

The N³-alkanesulfonyl derivatives of (3R)-aminopyrrolidine were obtainedby treating N¹-Boc-(3R)-aminopyrrolidine with propionylsulfonyl chlorideor cyclohexylmethylsulfonyl chloride (i-Pr₂NEt, CH₂Cl₂, 0° C.), anddeprotecting N-Boc group (CF₃CO₂H, CH₂Cl₂).

Derivatives of tetrahydro-3-thiophenamine-1,1-dioxide were preparedfollowing the protocol of Loev, B. J. Org. Chem. 1961, 26, 4394-9 byreacting 3-sulfolene with a requisite primary amine in methanol (cat.KOH, rt). N-Methyl-3-tetrahydrothiophene-amine-1,1-dioxide (TFA salt):¹H-NMR (DMSO-d₆): δ (ppm) 9.4 (br s, 2H), 4.0-3.8 (quin, 1H), 3.6-3.5(dd, 1H), 3.4-3.3 (m, 1H), 3.2-3.1 (m, 2H), 2.5 (s, 3H), 2.4 (m, 1H),2.1 (m, 1H).

(S)-1,1-Dioxo-tetrahydro-1λ⁶-thiophen-3-ylamine was prepared as follows:

1) N-Boc protection of (S)-3-tetrahydrothiophenamine (Dehmlow, E. V.;Westerheide, R. Synthesis 1992, 10, 947-9) by treating with (Boc)₂O inmethanol at room temperature for about 12 h; 2) oxidation by treatingwith mCPBA in dichloromethane to N-Boc protected(S)-1,1-dioxo-tetrahydro-1λ⁶-thiophen-3-ylamine at 0° C. for about 5 h;and 3) N-Boc deprotection of the sulfone derivative with TFA indichloromethane at room temperature for 1 h to the free amine which wasisolated as a TFA salt. (R)-1,1-dioxo-tetrahydro-1λ⁶-thiophen-3-ylaminewas prepared using the same method, but replacing the(S)-3-tetra-hydrothiophenamine with (R)-3-tetrahydrothiophenamine.

N-Methyl-tetrahydro-2H-thiopyran-4-amine-1,1-dioxide was prepared fromtetrahydro-4H-thiopyran-4-one: i) MeNH₂, NaBH₄; ii) (Boc)₂O, MeOH; iii)mCPBA, CH₂Cl₂, 0° C.; iv) CF₃CO₂H, CH₂Cl₂. (m/z): [M+H]⁺ calcd forC₆H₁₃NO₂S 164.07; found, 164.9. ¹H-NMR (CD₃OD; TFA salt): δ (ppm)3.4-3.1 (m, 5H), 2.7 (s, 3H), 2.4 (br d, 2H), 2.1 (br m, 2H).

Proline dimethylamide, isonipecotamide (piperidine-4-carboxamide), and1-(tetrahydro-2-furoyl)piperazine are available commercially, and werepurchased from commercial sources.

4-Piperidinol-dimethylcarbamate was prepared by reactingdimethylaminochloro-formate with N-Boc protected 4-piperidinol.

Preparation 1 Preparation of1-isopropyl-1,3-dihydro-2H-benzimidazol-2-one a. Preparation ofN-isopropyl-N-(2-nitrophenyl)amine

To a cold solution of 2-fluoro-nitrobenzene (31.8 g, 0.225 mol) inethanol (300 mL) cooled in an ice bath was added isopropylamine (54.0mL, 0.634 mol), followed by the addition of a solution of potassiumcarbonate (31.1 g, 0.225 mol) in water (120 mL). The mixture was stirredat 0° C. for 1 h, then refluxed for 6 h. The reaction was terminated bycooling the mixture to ambient temperature, and evaporating it underreduced pressure yielding an orange residue. The residue was partitionedbetween ethyl ether (800 mL) and a brine solution (300 mL). The organiclayer was dried and filtered, to provide the title intermediate (39 g)as an orange liquid. ¹H-NMR (CDCl₃, 300 MHz): δ (ppm) 8.06 (d, 1H), 7.30(t, 1H), 6.74 (d, 1H), 6.48 (t, 1H), 3.73 (hept, 1H), 1.20 (d, 6H).

b. Preparation of N-(2-aminophenyl)-N-isopropylamine

To a mixture of ethanol (600 mL) and 2 M sodium hydroxide solution (320mL) cooled in an ice bath was added Zn dust (59.5 g) slowly. Whilestirring the Zn slurry, N-isopropyl-N-(2-nitrophenyl)amine (41 g, 0.228mol) dissolved in ethanol (50 mL) was added. The mixture was stirred at0° C. for 30 min, then heated to 85° C. The mixture was stirred at 85°C. for about 12 h until the refluxing solution of the mixture became acolorless solution. The mixture was then cooled to 0° C. and filtered.The collected solid was rinsed with EtOAc (200 mL). The filtrate andrinsed solution were combined, and evaporated in vacuo to remove excessvolatile solvents. During the concentration, the mixture became palebrown/yellow. The aqueous concentrate was extracted with EtOAc (800 mL).The organic solution was concentrated to dryness, to provide the titleintermediate (33 g) as a brown-pink oil which was used in the next stepwithout further treatment. ¹H-NMR (CDCl₃, 300 MHz): δ (ppm) 6.73-6.5 (m,4H), 3.58-3.55 (hept, 1H), 1.2 (d, 6H).

c. Preparation of 1-isopropyl-1,3-dihydro-2H-benzimidazol-2-one

To a solution of the product of step (b),N-(2-aminophenyl)-N-isopropylamine (34 g, 0.226 mol), in tetrahydrofuran(500 mL) was added carbonyldiimidazole (36.7 g, 0.226 mol) as a solid.The mixture was stirred under an atmosphere of nitrogen gas at ambienttemperature for about 24 h. The mixture was concentrated in vacuo, and aresulting dark brown residue was distributed between EtOAc (700 mL) andbrine solution (300 mL). The organic layer was then washed with 1 Mphosphoric acid multiple times (˜3×300 mL) until the color of theorganic layer turned from dark brown to pale yellow. The organicsolution was evaporated to dryness to provide the title intermediate (34g) as a pale yellow oil which solidified slowly on standing. The purityof the material was assessed by ¹H-NMR which indicated no detectableimpurity: ¹H-NMR (CD₃OD, 300 MHz): δ (ppm) 7.2 (m, 1H), 7.0 (m, 3H), 4.6(hept, 1H), 1.46 (d, 6H). (m/z): [M+H]⁺ calcd for C₁₀H₁₂N₂O 177.09;found 177.2.

Anal. HPLC: retention time=2.7 min (99% purity): 1) column: Zorbax,Bonus-RP, 3.5 μm of particle size, 2.1×50 mm; 2) flow rate: 0.5 mL/min;3) isocratic condition (10% solvent B/90% solvent A) for 0 to 0.5 min;then linear gradient to 50% solvent B/50% solvent A over 5 min (solventA=98% water/2% MeCN/0.1% TFA; solvent B=90% MeCN/10% water/0.1% TFA).TLC analysis (silica gel plate): R_(f)=0.5 (CH₂Cl₂). Liquidchromatography mass spectrometry (LCMS) (m/z): [M+H]⁺ calcd forC₁₀H₁₂N₂O 177.09; found 177.3.

Preparation 2 Preparation of(1S,3R,5R)-3-amino-8-azabicyclo[3.2.1]octane-8-carboxylic acidtert-butyl ester a. Preparation of8-benzyl-8-azabicyclo[3.2.1]octan-3-one

Concentrated hydrochloric acid (30 mL) was added to a heterogeneoussolution of 2,5-dimethoxy tetrahydrofuran (82.2 g, 0.622 mol) in water(170 mL) while stirring. In a separate flask cooled to 0° C. (ice bath),concentrated hydrochloric acid (92 mL) was added slowly to a solution ofbenzyl amine (100 g, 0.933 mol) in water (350 mL). The2,5-dimethoxytetrahydrofuran solution was stirred for approximately 20min, diluted with water (250 mL), and then the benzyl amine solution wasadded, followed by the addition of a solution of 1,3-acetonedicarboxylicacid (100 g, 0.684 mol) in water (400 mL) and then the addition ofsodium hydrogen phosphate (44 g, 0.31 mol) in water (200 mL). The pH wasadjusted from pH 1 to pH ˜4.5 using 40% NaOH. The resulting solution wasstirred overnight. The solution was then acidified to pH 3 from pH 7.5using 50% hydrochloric acid, heated to 85° C. and stirred for 2 hours.The solution was cooled to room temperature, basified to pH 12 using 40%NaOH, and extracted with DCM (3×500 mL). The combined organic layerswere washed with brine, dried, filtered and concentrated under reducedpressure to produce the crude title intermediate as a viscous brown oil(52 g).

To a solution of the crude intermediate in methanol (1000 mL) was addeddi-tert-butyl dicarbonate (74.6 g, 0.342 mol) at 0° C. The solution wasallowed to warm to room temperature and stirred overnight. The methanolwas removed under reduced pressure and the resulting oil was dissolvedin dichloromethane (1000 mL). The intermediate was extracted into 1 MH₃PO₄ (1000 mL) and washed with dichloromethane (3×250 mL) The aqueouslayer was basified to pH 12 using aqueous NaOH, and extracted withdichloromethane (3×500 mL). The combined organic layers were dried,filtered and concentrated under reduced pressure to provide the titleintermediate as a viscous, light brown oil. ¹H-NMR (CDCl₃) δ (ppm)7.5-7.2 (m, 5H, C₆H₅), 3.7 (s, 2H, CH₂Ph), 3.45 (broad s, 2H, CH—NBn),2.7-2.6 (dd, 2H, CH₂CO), 2.2-2.1 (dd, 2H, CH₂CO), 2.1-2.0 (m, 2H,CH₂CH₂), 1.6 (m, 2H, CH₂CH₂). (m/z): [M+H]⁺ calcd for C₁₄H₁₇NO 216.14;found, 216.0.

b. Preparation of 3-oxo-8-azabicyclo[3.2.1]octane-8-carboxylic acidtert-butyl ester

To a solution of 8-benzyl-8-azabicyclo[3.2.1]octan-3-one (75 g, 0.348mol) in EtOAc (300 mL) was added a solution of di-tert-butyl dicarbonate(83.6 g, 0.383 mol, 1.1 eq) in EtOAc (300 mL). The resulting solutionand rinse (100 mL EtOAc) was added to a 1 L Parr hydrogenation vesselcontaining 23 g of palladium hydroxide (20 wt. % Pd, dry basis, oncarbon, ˜50% wet with water; e.g. Pearlman's catalyst) under a stream ofnitrogen. The reaction vessel was degassed (alternating vacuum and N₂five times) and pressurized to 60 psi of H₂ gas. The reaction solutionwas agitated for two days and recharged with H₂ as needed to keep the H₂pressure at 60 psi until the reaction was complete as monitored bysilica thin layer chromatography. The black solution was then filteredthrough a pad of Celite® and concentrated under reduced pressure toprovide the title intermediate as a viscous, yellow to orange oil. Itwas used in the next step without further treatment. ¹H NMR (CDCl₃) δ(ppm) 4.5 (broad, 2H, CH—NBoc), 2.7 (broad, 2H, CH₂CO), 2.4-2.3 (dd, 2H,CH₂CH₂), 2.1 (broad m, 2H, CH₂CO), 1.7-1.6 (dd, 2H, CH₂CH₂), 1.5 (s, 9H,(CH₃)₃COCON)).

c. Preparation of(1S,3R,5R)-3-amino-8-azabicyclo[3.2.1]octane-8-carboxylic acidtert-butyl ester

To a solution of the product of the previous step (75.4 g, 0.335 mol) inmethanol (1 L) was added ammonium formate (422.5 g, 6.7 mol), water (115mL) and 65 g of palladium on activated carbon (10% on dry basis, ˜50%wet with water; Degussa type E101NE/W) under a stream of N₂ whilestirring via mechanical stirrer. After 24 and 48 hours, additionalportions of ammonium formate (132 g, 2.1 mol) were added each time. Oncereaction progression ceased, as monitored by anal. HPLC, Celite® (>500g) was added and the resulting thick suspension was filtered and thenthe collected solid was rinsed with methanol (˜500 mL). The filtrateswere combined and concentrated under reduced pressure. The resultingcloudy, biphasic solution was then diluted with 1 M phosphoric acid to afinal volume of ˜1.5 to 2.0 L at pH 2 and washed with dichloromethane(3×700 mL). The aqueous layer was basified to pH 12 using 40% aq. NaOH,and extracted with dichloromethane (3×700 mL). The combined organiclayers were dried, filtered, and concentrated by rotary evaporation,then high-vacuum to provide the title intermediate (52 g), commonlyN-Boc-endo-3-aminotropane, as a white to pale yellow solid. The isomerratio of endo to exo amine of the product was >99:1 based on ¹H-NMRanalysis (>96% purity by analytical HPLC). ¹H NMR (CDCl₃) δ (ppm)4.2-4.0 (broad d, 2H, CHNBoc), 3.25 (t, 1H, CHNH₂), 2.1-2.05 (m, 4H),1.9 (m, 2H), 1.4 (s, 9H, (CH₃)₃OCON), 1.2-1.1 (broad, 2H). (m/z): [M+H]⁺calcd for C₁₂H₂₂N₂O₂ 227.18; found, 227.2. Analytical HPLC (isocraticmethod; 2:98 (A:B) to 90:10 (A:B) over 5 min): retention time=3.68 min.

Example 1 Synthesis of4-(tetrahydrofuran-2-carbonyl)piperazine-1-carboxylic acid3-{(1S,3R,5R)-3-[(3-isopropyl-2-oxo-2,3-dihydrobenzoimidazole-1-carbonyl)amino]-8-aza-bicyclo[3.2.1]oct-8-yl}propylester

a. Preparation of(1S,3R,5R)-3-[(3-isopropyl-2-oxo-2,3-dihydrobenzoimidazole-1-carbonyl)amino]-8-azabicyclo[3.2.1]octane-8-carboxylicacid tert-butyl ester

To a cold suspension of sodium hydride (9.25 g; 231.4 mmol; 60%dispersion in mineral oil) in dry THF (1000 L) in an ice bath was addedthe product of Preparation 1,1-isopropyl-1,3-dihydro-2H-benzimidazol-2-one (27.2 g, 154.2 mmol), inTHF (50 mL) under nitrogen atmosphere. The mixture was stirred at ˜0-5°C. for 30 min, then 4-nitrophenyl chloroformate (34.2 g, 170 mmol) inTHF (50 mL) was added. The mixture was stirred overnight while allowingthe mixture to gradually warm to ambient temperature. To the activatedester formed was then added(1S,3R,5R)-3-amino-8-azabicyclo[3.2.1]octane-8-carboxylic acidtert-butyl ester (36.7 g, 162 mmol) in THF (50 mL). The mixture wasstirred at ambient temperature for about 12 h, and at about 75° C. forabout 3 h, at which time an LCMS of the reaction sample indicatedcompletion of the coupling reaction. The mixture was concentrated invacuo, dissolved in dichloromethane (1 L), and washed with first 1MH₃PO₄, and then saturated NaHCO₃ solution. After drying, the organicsolution was evaporated to provide the title intermediate as a paleyellow residue that was used in the next step without further treatment.

b. Preparation ofN-[(1S,3R,5R)-3-isopropyl-2-oxo-2,3-dihydrobenzoimidazole-1-carboxylicacid (8-azabicyclo[3.2.1]oct-3-yl)amide as a trifluoroacetate salt

To a cold solution of(1S,3R,5R)-3-[(3-isopropyl-2-oxo-2,3-dihydrobenzo-imidazole-1-carbonyl)amino]-8-azabicyclo[3.2.1]octane-8-carboxylicacid tert-butyl ester (the product of the previous step) indichloromethane (200 mL) in an ice bath was added trifluoroacetic acid(200 mL). The mixture was stirred for about 30 min at ˜5° C., and atroom temperature for about 1 h. After evaporation of the mixture, ethylether (˜500 mL) was added to the oily residue, causing solidification ofthe residue. The precipitate was collected, rinsed with copious amountsof ethyl ether, and dried in vacuo, to provide the title intermediate(47 g) as a TFA salt. The title intermediate is also commonly referredto asendo-N-(8-azabicyclo[3.2.1]oct-3-yl)-3-isopropyl-2-oxo-2,3-dihydrobenzimidazole-1-carboxamide.

c. Preparation ofN-[(1S,3R,5R)-3-isopropyl-2-oxo-2,3-dihydrobenzoimidazole-1-carboxylicacid (8-azabicyclo[3.2.1]oct-3-yl)amide (freebase)

To a suspension of the product of the previous step (15 g, 33.9 mmol) indichloromethane (500 mL) was added water (500 mL).N,N-diisopropylethylamine (˜20 mL) was added to the reaction mixture tobring the aqueous layer to a pH of 8-9. The layers were separated,retaining the organic layer. The aqueous layer was extracted a secondtime with dichloromethane (100 mL). The resulting extracts werecombined, and then washed with brine. After drying over Na₂SO₄ andfiltration, removal of the solvent yielded the title compound (9.7 g) asa freebase as a yellow powder. ¹H NMR (DMSO-d₆): 1.48 (d, 6H), 1.40-2.00(m, 8H), 3.53 (m, 2H), 4.07 (m, 1H), 4.69 (septet, 1H), 7.21 (m, 2H),7.45 (d, 1H), 8.08 (d, 1H), 9.31 (d, 1H). (m/z): [M+H]⁺ calcd forC₁₈H₂₄N₄O₂ 329.20; found 329.2. Analytical HPLC: (2-50% MeCN/H₂O over 6min) retention time=3.67 min.

d. Preparation of3-chloropropyl-4-(tetrahydrofuran-2-ylcarbonyl)piperazine-1-carboxylate

To a 0° C. solution of 1-(tetrahydrofuran-2-ylcarbonyl)piperazine (202mg, 1.1 mmol) in dichloromethane (5 mL) was added 3-chloropropylchloroformate (133 μL, 1.1 mmol) followed by N,N-diisopropylethylamine(192 μL, 1.1 mmol). The reaction was allowed to reach room temperatureover 2 h, at which time the reaction was evaporated to yield the titlecompound as a wheat colored oil that was used without furtherpurification.

e. Synthesis of 4-(tetrahydrofuran-2-carbonyl)piperazine-1-carboxylicacid3-{(1S,3R,5R)-3-[(3-isopropyl-2-oxo-2,3-dihydrobenzoimidazole-1-carbonyl)amino]-8-aza-bicyclo[3.2.1]oct-8-yl}propylester

3-Chloropropyl-4-(tetrahydrofuran-2-ylcarbonyl)piperazine-1-carboxylate(335 mg, 1.1 mmol) was dissolved in dimethylformamide (5.0 mL) and addedto the solid freebase product of step (c) (118 mg, 0.36 mmol) and NaI(164 mg, 0.72 mmol). N,N-diisopropylethylamine (64 μL, 0.36 mmol) wasadded and the mixture stirred at 90° C. overnight. Volatiles wereremoved and purification via prep HPLC (reverse phase) was accomplishedon a gradient of 15-45% over 50 min; flow rate 20 mL/min to provide thetitle compound as a white solid as a TFA salt (45 mg). (m/z): [M+H]⁺calcd for C₃₁H₄₄N₆O₆ 597.33; found 597.1. Analytical HPLC: (5-65%MeCN/H₂O over 4 min) retention time=2.58.

Example 2 Synthesis of 4-(2-hydroxyethyl)piperazine-1-carboxylic acid3-{(1S,3R,5R)-3-[(3-isopropyl-2-oxo-2,3-dihydrobenzoimidazole-1-carbonyl)amino]-8-azabicyclo-[3.2.1]oct-8-yl}propylester

Using the processes described in Example 1, except in step (d) replacing1-(tetrahydrofuran-2-ylcarbonyl)piperazine with 2-piperazin-1-ylethanol,the title compound (13.8 mg) was prepared as a TFA salt. (m/z): [M+H]⁺calcd for C₂₈H₄₂N₆O₅ 543.32; obsd. 543.5.

Examples 3-12

Using the processes described in Example 1, except in step (d) replacing1-(tetrahydrofuran-2-ylcarbonyl)piperazine with the appropriatereagents, the following compounds of Examples 3-12 were prepared.

Calc. Obs. Example —NR³R⁴ Formula [M + H]⁺ [M + H]⁺ 3

C₂₈H₄₀N₆O₅ 541.31 541.2 4

C₃₀H₄₄N₆O₆ 585.33 585.2 5

C₂₈H₄₀N₆O₅ 541.31 541.2 6

C₂₆H₃₇N₅O₆S 548.25 548.2 7

C₂₇H₃₉N₅O₆S 562.26 562.2 8

C₂₇H₃₈N₆O₅ 527.29 527.2 9

C₂₉H₄₂N₆O₅ 555.32 555.2 10

C₂₇H₄₀N₆O₅ 529.31 529.2 11

C₂₉H₄₃N₇O₅ 570.30 570.4 12

C₂₈H₄₃N₇O₆S 606.30 606.2

Example 13

Alternative synthesis of 1,1-dioxo-1λ⁶-thiomorpholine-4-carboxylic acid3-{(1S,3R,5R)-3-[(3-isopropyl-2-oxo-2,3-dihydrobenzoimidazole-1-carbonyl)amino]-8-azabicyclo[3.2.1]oct-8-yl}propylester

a. Preparation of(1S,3R,5R)-3-[(3-isopropyl-2-oxo-2,3-dihydrobenzoimidazole-1-carbonyl)amino]-8-azabicyclo[3.2.1]octane-8-carboxylicacid tert-butyl ester

To a 500 mL reaction flask containing1-isopropyl-1,3-dihydro-2H-benzimidazol-2-one (17.6 g, 100 mmol) and4-nitrophenyl chloroformate (20.2 g, 100 mmol) under nitrogen atmospherewas added dichloromethane (350 mL) and then triethylamine (30.5 mL, 220mmol) was added slowly. The solution was stirred for 15 min and then(1S,3R,5R)-3-amino-8-azabicyclo[3.2.1]octane-8-carboxylic acidtert-butyl ester (22.6 g, 100 mmol) was added. The reaction was allowedto stir overnight at room temperature. The reaction mixture was washedwith aqueous saturated sodium bicarbonate (2×200 mL). Dichloromethanewas removed by distillation and MTBE (350 mL) was added. The MTBEsolution was washed with 1N phosphoric acid (2×200 mL), saturated sodiumbicarbonate (200 mL), and water (200 mL). The organic layer was driedover anyhydrous sodium sulfate (40 g) and filtered, and then the solventwas removed by distillation to yield the title intermediate as a tansolid (35.7 g, 83% yield).

b. Preparation ofN-[(1S,3R,5R)-3-isopropyl-2-oxo-2,3-dihydrobenzoimidazole-1-carboxylicacid (8-azabicyclo[3.2.1]oct-3-yl)amide trifluoroacetate salt

In a 500 mL flask,(1S,3R,5R)-3-[(3-isopropyl-2-oxo-2,3-dihydrobenzoimidazole-1-carbonyl)amino]-8-azabicyclo[3.2.1]octane-8-carboxylicacid tert-butyl ester (21.4 g, 50 mmol) was dissolved in dichloromethane(200 mL). Trifluoroacetic acid (37 mL, 500 mmol) was added and thereaction mixture was allowed to stir at room temperature for 3 h. Thereaction mixture was washed with water (2×100 mL). The solvent in theorganic layer was removed by distillation and the crude product residuetriturated by addition of MTBE (200 mL). After stirring for 1 h at roomtemperature, the solids were isolated by filtration, washed with MTBE(2×25 mL) and dried under vacuum to provide the title intermediate (21.0g, 97% yield).

c. Preparation of 1,1-dioxo-1λ⁶-thiomorpholine-4-carboxylic acid3-chloropropyl ester

In a 500 mL flask, thiomorpholine dioxide (13.5 g, 100 mmol) wasdissolved in dichloromethane (150 mL) at room temperature andN,N-diisopropylethylamine (19.2 mL, 110 mmol) was added. After stirringat room temperature for 10 min, the reaction mixture was cooled in anice bath to approximately 5° C. To the reaction mixture, was added1-chloro-3-chloromethoxypropane (11.8 mL, 100 mmol) via addition funnelat a rate which maintained the reaction temperature below 10° C. Whenthe addition was complete, the reaction mixture was allowed to warm toroom temperature. The reaction mixture was washed with water (2×100 mL),and the organic phase dried over anhydrous sodium sulfate (25 g). Afterfiltration, the solvent was removed by distillation to yield the titlecompound as an oily solid that solidified upon standing (24.0 g, 94%yield).

e. Synthesis of 1,1-dioxo-1λ⁶-thiomorpholine-4-carboxylic acid3-{(1S,3R,5R)-3-[(3-isopropyl-2-oxo-2,3-dihydrobenzoimidazole-1-carbonyl)amino]-8-azabicyclo[3.2.1]oct-8-yl}propylester

To a solution ofN-[(1S,3R,5R)-3-isopropyl-2-oxo-2,3-dihydrobenzoimidazole-1-carboxylicacid (8-azabicyclo[3.2.1]oct-3-yl)amide trifluoroacetate salt (8.8 g, 20mmol) in dichloromethane (100 mL) was added water (100 mL). The pH ofthe aqueous layer was adjusted to ˜12 to provide the freebase of thesalt. The organic layer was separated, dried over sodium sulfate and thesolvent was removed by distillation. The freebase was dissolved inN-methyl-2-pyrrolidone (100 mL) and the solution transferred to a 250 mLflask containing 1,1-dioxo-1λ⁶-thiomorpholine-4-carboxylic acid3-chloropropyl ester (7.2 g, 28 mmol) and NaI (3.0 g, 20 mmol).N,N-diisopropylethylamine (4.2 mL, 24 mmol) was added and the reactionmixture was heated to 50° C. for 18 h. The solvent was removed bydistillation. The crude product residue was dissolved in EtOAc (200 mL),washed with water (2×50 mL), and dried over sodium sulfate (10 g). Thesolvent was removed by distillation to afford crude product residue (˜12g).

The crude product residue was purified by preparative HPLC on a 2″column; packing: base-deactivated silica (BDS), flow rate: 200 mL/min;eluent A: 0.1% TFA in water; eluent B: 90% acetonitrile/10% 0.1% TFA inwater; gradient (time, % B): (0, 5); (25, 30); (35, 80); (45, 80); (50,5); (60, 5). The product was isolated by lyophilization of the purefractions to provide the title compound (3.4 g, 26% yield). ¹H-NMR(DMSO-d₆) δ (ppm): 9.35 (d, 1H), 8.07 (d, 1H), 7.46 (d, 1H), 7.22 (t,1H), 7.16 (t, 1H), 4.69 (septet, 1H), 4.20-4.00 (m, 3H), 4.12 (t, 2H),3.90-3.70 (m, 4H), 3.70 (t, 2H), 3.25-3.05 (m, 4H), 2.5-2.0 (m, 8H),2.15 (dt, 2H), 1.49 (d, 6H). (m/z): [M+H]⁺ calcd for C₂₆H₃₇N₅O₆S 548.2;found, 548.4

Assay 1: Radioligand Binding Assay on 5-HT_(4(C)) Human Receptors

a. Membrane Preparation 5-HT_(4(c))

HEK-293 (human embryonic kidney) cells stably-transfected with human5-HT_(4(C)) receptor cDNA (Bmax=˜6.0 pmol/mg protein, as determinedusing [³H]-GR113808 membrane radioligand binding assay) were grown inT-225 flasks in Dulbecco's Modified Eagles Medium (DMEM) containing4,500 mg/L D-glucose and pyridoxine hydrochloride (GIBCO-InvitrogenCorp., Carlsbad Calif.: Cat #11965) supplemented with 10% fetal bovineserum (FBS) (GIBCO-Invitrogen Corp.: Cat #10437), 2 mM L-glutamine and(100 units) penicillin-(100 μg) streptomycin/ml (GIBCO-Invitrogen Corp.:Cat #15140) in a 5% CO₂, humidified incubator at 37° C. Cells were grownunder continuous selection pressure by the addition of 800 μg/mLgeneticin (GIBCO-Invitrogen Corp.: Cat # 10131) to the medium.

Cells were grown to roughly 60-80% confluency (<35 subculture passages).At 20-22 hours prior to harvesting, cells were washed twice and fed withserum-free DMEM. All steps of the membrane preparation were performed onice. The cell monolayer was lifted by gentle mechanical agitation andtrituration with a 25 mL pipette. Cells were collected by centrifugationat 1000 rpm (5 min).

For the membrane preparation, cell pellets were resuspended in ice-cold50 mM 4-(2-hydroxyethyl)-1-piperazineethanesulphonic acid (HEPES), pH7.4 (membrane preparation buffer) (40 mL/total cell yield from 30-40T225 flasks) and homogenized using a polytron disrupter (setting 19,2×10 s) on ice. The resultant homogenates were centrifuged at 1200 g for5 min at 4° C. The pellet was discarded and the supernatant centrifugedat 40,000 g (20 min). The pellet was washed once by resuspension withmembrane preparation buffer and centrifugation at 40,000 g (20 min). Thefinal pellet was resuspended in 50 mM HEPES, pH 7.4 (assay buffer)(equivalent I T225 flask/1 mL). Protein concentration of the membranesuspension was determined by the method of Bradford (Bradford, 1976).Membranes were stored frozen in aliquots at −80° C.

b. Radioligand Binding Assays

Radioligand binding assays were performed in 11.1 mL 96-deep wellpolypropylene assay plates (Axygen) in a total assay volume of 400 μLcontaining 2 μg membrane protein in 50 mM HEPES pH 7.4, containing0.025% bovine serum albumin (BSA). Saturation binding studies fordetermination of K_(d) values of the radioligand were performed using[³H]-GR113808 (Amersham Inc., Bucks, UK: Cat #TRK944; specific activity˜82 Ci/mmol) at 8-12 different concentrations ranging from 0.001 nM-5.0nM. Displacement assays for determination of pK_(i) values of compoundswere performed with [³H]-GR113808 at 0.15 nM and eleven differentconcentrations of compound ranging from 10 pM-100 μM.

Test compounds were received as 10 mM stock solutions in DMSO anddiluted to 400 μM into 50 mM HEPES pH 7.4 at 25° C., containing 0.1%BSA, and serial dilutions (1:5) then made in the same buffer.Non-specific binding was determined in the presence of 1 μM unlabeledGR113808. Assays were incubated for 60 min at room temperature, and thenthe binding reactions were terminated by rapid filtration over 96-wellGF/B glass fiber filter plates (Packard BioScience Co., Meriden, Conn.)presoaked in 0.3% polyethyleneimine. Filter plates were washed threetimes with filtration buffer (ice-cold 50 mM HEPES, pH7.4) to removeunbound radioactivity. Plates were dried, 35 μL Microscint-20 liquidscintillation fluid (Packard BioScience Co., Meriden, Conn.) was addedto each well and plates were counted in a Packard Topcount liquidscintillation counter (Packard BioScience Co., Meriden, Conn.).

Binding data were analyzed by nonlinear regression analysis with theGraphPad Prism Software package (GraphPad Software, Inc., San Diego,Calif.) using the 3-parameter model for one-site competition. The BOTTOM(curve minimum) was fixed to the value for nonspecific binding, asdetermined in the presence of 1 μM GR113808. K_(i) values for testcompounds were calculated, in Prism, from the best-fit IC₅₀ values, andthe K_(d) value of the radioligand, using the Cheng-Prusoff equation(Cheng and Prusoff, Biochemical Pharmacology, 1973, 22, 3099-108):K_(i)=IC₅₀/(1+[L]/K_(d)) where [L]=concentration [³H]-GR113808. Resultsare expressed as the negative decadic logarithm of the K_(i) values,pK_(i).

Test compounds having a higher pK_(i) value in this assay have a higherbinding affinity for the 5-HT₄ receptor. The compounds of the inventionwhich were tested in this assay had a pK_(i) value ranging from about7.0 to about 9.0, typically ranging from about 7.5 to about 8.5. Forexample, the compound of Example 1 exhibited a pK_(i) value of 7.9 inthis assay.

Assay 2: Radioligand Binding Assay on 5-HT_(3A) Human Receptors:Determination of Receptor Subtype Selectivity

a. Membrane Preparation 5-HT_(3A)

HEK-293 (human embryonic kidney) cells stably-transfected with human5-HT_(3A) receptor cDNA were obtained from Dr. Michael Bruess(University of Bonn, GDR) (Bmax=˜9.0 μmol/mg protein, as determinedusing [³H]-GR65630 membrane radioligand binding assay). Cells were grownin T-225 flasks or cell factories in 50% Dulbecco's Modified EaglesMedium (DMEM) (GIBCO-Invitrogen Corp., Carlsbad, Calif.: Cat #11965) and50% Ham's F12 (GIBCO-Invitrogen Corp.: Cat #11765) supplemented with 10%heat inactivated fetal bovine serum (FBS) (Hyclone, Logan, Utah: Cat#SH30070.03) and (50 units) penicillin-(50 μg) streptomycin/ml(GIBCO-Invitrogen Corp.: Cat #15140) in a 5% CO₂, humidified incubatorat 37° C.

Cells were grown to roughly 70-80% confluency (<35 subculture passages).All steps of the membrane preparation were performed on ice. To harvestthe cells, the media was aspirated and cells were rinsed with Ca²⁺,Mg²⁺-free Dulbecco's phosphate buffered saline (dPBS). The cellmonolayer was lifted by gentle mechanical agitation. Cells werecollected by centrifugation at 1000 rpm (5 min). Subsequent steps of themembrane preparation followed the protocol described above for themembranes expressing 5-HT_(4(c)) receptors.

b. Radioligand Binding Assays

Radioligand binding assays were performed in 96-well polypropylene assayplates in a total assay volume of 200 μL containing 1.5-2 μg membraneprotein in 50 mM HEPES pH 7.4, containing 0.025% BSA assay buffer.Saturation binding studies for determination of K_(d) values of theradioligand were performed using [³H]-GR65630 (PerkinElmer Life SciencesInc., Boston, Mass.: Cat #NET1011, specific activity ˜85 Ci/mmol) attwelve different concentrations ranging from 0.005 nM to 20 nM.Displacement assays for determination of pK_(i) values of compounds wereperformed with [³H]-GR65630 at 0.50 nM and eleven differentconcentrations of compound ranging from 10 pM to 100 μM. Compounds werereceived as 10 mM stock solutions in DMSO (see section 3.1), diluted to400 μM into 50 mM HEPES pH 7.4 at 25° C., containing 0.1% BSA, andserial (1:5) dilutions then made in the same buffer. Non-specificbinding was determined in the presence of 10 μM unlabeled MDL72222.Assays were incubated for 60 min at room temperature, then the bindingreactions were terminated by rapid filtration over 96-well GF/B glassfiber filter plates (Packard BioScience Co., Meriden, Conn.) presoakedin 0.3% polyethyleneimine. Filter plates were washed three times withfiltration buffer (ice-cold 50 mM HEPES, pH7.4) to remove unboundradioactivity. Plates were dried, 35 μL Microscint-20 liquidscintillation fluid (Packard BioScience Co., Meriden, Conn.) was addedto each well and plates were counted in a Packard Topcount liquidscintillation counter (Packard BioScience Co., Meriden, Conn.).

Binding data were analyzed using the non-linear regression proceduredescribed above to determine K_(i) values. The BOTTOM (curve minimum)was fixed to the value for nonspecific binding, as determined in thepresence of 10 μM MDL72222. The quantity [L] in the Cheng-Prusoffequation was defined as the concentration [³H]-GR65630.

Selectivity for the 5-HT₄ receptor subtype with respect to the 5-HT₃receptor subtype was calculated as the ratioK_(i)(5-HT_(3A))/K_(i)(5-HT_(4(c))). The compounds of the inventionwhich were tested in this assay had a 5-HT₄/5-HT₃ receptor subtypeselectivity ranging from about 10 to about 950, typically ranging fromabout 50 to about 500. For example, the compound of Example I exhibiteda subtype selectivity of 160.

Assay 3: Whole-Cell cAMP Accumulation Flashplate Assay with HEK-293Cells Expressing Human 5-HT_(4(c)) Receptors

In this assay, the functional potency of a test compound was determinedby measuring the amount of cyclic AMP produced when HEK-293 cellsexpressing 5-HT₄ receptors were contacted with different concentrationsof test compound.

a. Cell Culture

HEK-293 (human embryonic kidney) cells stably-transfected with clonedhuman 5-HT_(4(c)) receptor cDNA were prepared expressing the receptor attwo different densities: (1) at a density of about 0.5-0.6 pmol/mgprotein, as determined using a [³H]-GR113808 membrane radioligandbinding assay, and (2) at a density of about 6.0 pmol/mg protein. Thecells were grown in T-225 flasks in Dulbecco's Modified Eagles Medium(DMEM) containing 4,500 mg/L D-glucose (GIBCO-Invitrogen Corp.: Cat#11965) supplemented with 10% fetal bovine serum (FBS) (GIBCO-InvitrogenCorp.: Cat #10437) and (100 units) penicillin-(100 μg) streptomycin/ml(GIBCO-Invitrogen Corp.: Cat #15140) in a 5% CO₂, humidified incubatorat 37° C. Cells were grown under continuous selection pressure by theaddition of geneticin (800 μg/mL: GIBCO-Invitrogen Corp.: Cat #10131) tothe medium.

b. Cell Preparation

Cells were grown to roughly 60-80% confluency. Twenty to twenty-twohours prior to assay, cells were washed twice, and fed, with serum-freeDMEM containing 4,500 mg/L D-glucose (GIBCO-Invitrogen Corp.: Cat#11965). To harvest the cells, the media was aspirated and 10 mL Versene(GIBCO-Invitrogen Corp.: Cat #15040) was added to each T-225 flask.Cells were incubated for 5 min at RT and then dislodged from the flaskby mechanical agitation. The cell suspension was transferred to acentrifuge tube containing an equal volume of pre-warmed (37° C.) dPBSand centrifuged for 5 min at 1000 rpm. The supernatant was discarded andthe pellet was re-suspended in pre-warmed (37° C.) stimulation buffer(10 mL equivalent per 2-3 T-225 flasks). This time was noted and markedas time zero. The cells were counted with a Coulter counter (count above8 μm, flask yield was 1-2×10⁷ cells/flask). Cells were resuspended at aconcentration of 5×10⁵ cells/ml in pre-warned (37° C.) stimulationbuffer (as provided in the flashplate kit) and preincubated at 37° C.for 10 min.

cAMP assays were performed in a radioimmunoassay format using theFlashplate Adenylyl Cyclase Activation Assay System with ¹²⁵I-cAMP(SMP004B, PerkinElmer Life Sciences Inc., Boston, Mass.), according tothe manufacturer's instructions.

Cells were grown and prepared as described above. Final cellconcentrations in the assay were 25×10³ cells/well and the final assayvolume was 100 μL. Test compounds were received as 10 mM stock solutionsin DMSO, diluted to 400 μM into 50 mM HEPES pH 7.4 at 25° C., containing0.1% BSA, and serial (1:5) dilutions then made in the same buffer.Cyclic AMP accumulation assays were performed with 11 differentconcentrations of compound ranging from 10 μM to 100 μM (final assayconcentrations). A 5-HT concentration-response curve (10 μM to 100 μM)was included on every plate. The cells were incubated, with shaking, at37° C. for 15 min and the reaction terminated by addition of 100 μl ofice-cold detection buffer (as provided in the flashplate kit) to eachwell. The plates were sealed and incubated at 4° C. overnight. Boundradioactivity was quantified by scintillation proximity spectroscopyusing the Topcount (Packard BioScience Co., Meriden, Conn.).

The amount of cAMP produced per mL of reaction was extrapolated from thecAMP standard curve, according to the instructions provided in themanufacturer's user manual. Data were analyzed by nonlinear regressionanalysis with the GraphPad Prism Software package using the 3-parametersigmoidal dose-response model (slope constrained to unity). Potency dataare reported as pEC₅₀ values, the negative decadic logarithm of the EC₅₀value, where EC₅₀ is the effective concentration for a 50% maximalresponse.

Test compounds exhibiting a higher pEC₅₀ value in this assay have ahigher potency for agonizing the 5-HT₄ receptor. The compounds of theinvention which were tested in this assay using cell line (1) having adensity of about 0.5-0.6 μmol/mg protein, had a pEC₅₀ value in the rangeof about 7.5 to about 9.0, typically in the range of about 8.0 to about9.0. For example, the compound of Example 1 had a pEC₅₀ value of 8.4.

Assay 4: In Vitro Voltage Clamp Assay of Inhibition of Potassium IonCurrent in Whole Cells Expressing the hERG Cardiac Potassium Channel

CHO-K1 cells stably transfected with hERG cDNA were obtained from GailRobertson at the University of Wisconsin. Cells were held in cryogenicstorage until needed. Cells were expanded and passaged in Dulbecco'sModified Eagles Medium/F12 supplemented with 10% fetal bovine serum and200 μg/mL geneticin. Cells were seeded onto poly-D-lysine (100 μg/mL)coated glass coverslips, in 35 mm² dishes (containing 2 mL medium) at adensity that enabled isolated cells to be selected for whole cellvoltage-clamp studies. The dishes were maintained in a humidified, 5%CO₂ environment at 37° C.

Extracellular solution was prepared at least every 7 days and stored at4° C. when not in use. The extracellular solution contained (mM): NaCl(137), KCl (4), CaCl₂ (1.8), MgCl₂ (1), Glucose (10),4-(2-hydroxyethyl)-1-piperazineethanesulphonic acid (HEPES) (10), pH 7.4with NaOH. The extracellular solution, in the absence or presence oftest compound, was contained in reservoirs, from which it flowed intothe recording chamber at approximately 0.5 mL/min. The intracellularsolution was prepared, aliquoted and stored at −20° C. until the day ofuse. The intracellular solution contained (mM): KCl (130), MgCl₂ (1),ethylene glycol-bis(beta-aminoethyl ether) N,N,N′,N′-tetra acetic acidsalt (EGTA) (5), MgATP (5),4-(2-hydroxyethyl)-1-piperazineethanesulphonic acid (HEPES) (10), pH 7.2with KOH. All experiments were performed at room temperature (20-22°C.).

The coverslips on which the cells were seeded were transferred to arecording chamber and perfused continuously. Gigaohm seals were formedbetween the cell and the patch electrode. Once a stable patch wasachieved, recording commenced in the voltage clamp mode, with theinitial holding potential at −80 mV. After a stable whole-cell currentwas achieved, the cells were exposed to test compound. The standardvoltage protocol was: step from the holding potential of −80 mV to +20mV for 4.8 sec, repolarize to −50 mV for 5 sec and then return to theoriginal holding potential (−80 mV). This voltage protocol was run onceevery 15 sec (0.067 Hz). Peak current amplitudes during therepolarization phase were determined using pClamp software. Testcompounds at a concentration of 3 μM were perfused over the cells for 5minutes, followed by a 5-minute washout period in the absence ofcompound. Finally a positive control (cisapride, 20 nM) was added to theperfusate to test the function of the cell. The step from −80 mV to +20mV activates the hERG channel, resulting in an outward current. The stepback to −50 mV results in an outward tail current, as the channelrecovers from inactivation and deactivates.

Peak current amplitudes during the repolarization phase were determinedusing pCLAMP software. The control and test article data were exportedto Origin® (OriginLab Corp., Northampton Mass.) where the individualcurrent amplitudes were normalized to the initial current amplitude inthe absence of compound. The normalized current means and standarderrors for each condition were calculated and plotted versus the timecourse of the experiment.

Comparisons were made between the observed K⁺ current inhibitions afterthe five-minute exposure to either the test article or vehicle control(usually 0.3% DMSO). Statistical comparisons between experimental groupswere performed using a two-population, independent t-test (MicrocalOrigin v. 6.0). Differences were considered significant at p<0.05.

The smaller the percentage inhibition of the potassium ion current inthis assay, the smaller the potential for test compounds to change thepattern of cardiac repolarization when used as therapeutic agents. Thecompounds of the invention which were tested in this assay at aconcentration of 3 μM typically exhibited an inhibition of the potassiumion current of less than about 40%, more typically, less than about 25%.For example, the compound of Example 1 exhibited an inhibition of about9% in this assay.

Assay 5: In Vitro Model of Oral Bioavailability: Caco-2 Permeation Assay

The Caco-2 permeation assay was performed to model the ability of testcompounds to pass through the intestine and get into the blood streamafter oral administration. The rate at which test compounds in solutionpermeate a cell monolayer designed to mimic the tight junction of humansmall intestinal monolayers was determined.

Caco-2 (colon, adenocarcinoma; human) cells were obtained from ATCC(American Type Culture Collection; Rockville, Md.). For the permeationstudy, cells were seeded at a density of 63,000 cells/cm² on pre-wettedtranswells polycarbonate filters (Costar; Cambridge, Mass.). A cellmonolayer was formed after 21 days in culture. Following cell culture inthe transwell plate, the membrane containing the cell monolayer wasdetached from the transwell plate and inserted into the diffusionchamber (Costar; Cambridge, Mass.). The diffusion chamber was insertedinto the heating block which was equipped with circulating external,thermostatically regulated 37° C. water for temperature control. The airmanifold delivered 95% O₂/5% CO₂ to each half of a diffusion chamber andcreated a laminar flow pattern across the cell monolayer, which waseffective in reducing the unstirred boundary layer.

The permeation study was performed with test compound concentrations at100 μM and with ¹⁴C-mannitol to monitor the integrity of the monolayer.All experiments were conducted at 37° C. for 60 min. Samples were takenat 0, 30 and 60 min from both the donor and receiver sides of thechamber. Samples were analyzed by HPLC or liquid scintillation countingfor test compound and mannitol concentrations. The permeationcoefficient (K_(p)) in cm/sec was calculated.

In this assay, a K_(p) value greater than about 10×10⁻⁶ cm/sec isconsidered indicative of favorable bioavailability. The compounds of theinvention that were tested in this assay typically exhibited K_(p)values of between about 20×10⁻⁶ cm/sec and about 60×10⁻⁶ cm/sec, moretypically between about 30×10⁻⁶ cm/sec and about 60×10⁻⁶ cm/sec. Forexample, the compound of Example I exhibited a K_(p) value of 60×10⁻⁶cm/sec.

Assay 6: Pharmacokinetic Study in the Rat

Aqueous solution formulations of test compounds were prepared in 0.1%lactic acid at a pH of between about 5 and about 6. Male Sprague-Dawleyrats (CD strain, Charles River Laboratories, Wilmington, Mass.) weredosed with test compounds via intravenous administration (IV) at a doseof 2.5 mg/kg or by oral gavage (PO) at a dose of 5 mg/kg. The dosingvolume was 1 mL/kg for IV and 2 mL/kg for PO administration. Serialblood samples were collected from animals pre-dose, and at 2 (IV only),5, 15, and 30 min, and at 1, 2, 4, 8, and 24 hours post-dose.Concentrations of test compounds in blood plasma were determined byliquid chromatography-mass spectrometry analysis (LC-MS/MS) (MDS SCIEX,API 4000, Applied Biosystems, Foster City, Calif.) with a lower limit ofquantitation of 1 ng/mL.

Standard pharmacokinetic parameters were assessed by non-compartmentalanalysis (Model 201 for IV and Model 200 for PO) using WinNonlin(Version 4.0.1, Pharsight, Mountain View, Calif.). The maximum in thecurve of test compound concentration in blood plasma vs. time is denotedC_(max). The area under the concentration vs. time curve from the timeof dosing to the last measurable concentration (AUC(O-t)) was calculatedby the linear trapezoidal rule. Oral bioavailability (F(%)), i.e. thedose-normalized ratio of AUC(O-t) for PO administration to AUC(O-t) forIV administration, can be calculated as:F(%)=AUC _(PO) /AUC _(IV)×Dose_(IV)/Dose_(PO)×100%

Test compounds which exhibit larger values of the parameters C_(max),AUC(0−t), and F(%) in this assay are expected to have greaterbioavailability when administered orally. The compounds of the inventionthat were tested in this assay had C_(max) values between about 0.15 toabout 0.35 μg/mL and AUC(0−t) values between about 0.5 to about 1.1μg·hr/mL. In particular, the compound of Example I had the followingvalues: C_(max) of 0.32 μg/mL; AUC(O-t) of 0.97 μg-hr/mL; and oralbioavailability F(%) of 55%.

While the present invention has been described with reference to thespecific embodiments thereof, it should be understood by those skilledin the art that various changes may be made and equivalents may besubstituted without departing from the true spirit and scope of theinvention. In addition, many modifications may be made to adapt aparticular situation, material, composition of matter, process, processstep or steps, to the objective, spirit and scope of the presentinvention. All such modifications are intended to be within the scope ofthe claims appended hereto. Additionally, all publications, patents, andpatent documents cited hereinabove are incorporated by reference hereinin full, as though individually incorporated by reference.

1-20. (canceled)
 21. A method of treating a disorder of reduced motilityof the gastrointestinal tract in a mammal, the method comprisingadministering to the mammal, a therapeutically effective amount of apharmaceutical composition comprising a pharmaceutically-acceptablecarrier and a compound of formula (I):

wherein: R¹ is halo or C₁₋₃alkyl, wherein C₁₋₃alkyl is optionallysubstituted with hydroxy or halo; R² is hydrogen or C₁₋₃alkyl, whereinC₁₋₃alkyl is optionally substituted with hydroxy; R³ is C₁₋₃alkyl orhydrogen; R⁴ is —(CH₂)₁₋₃C(O)NR^(a)R^(b),

or R³ and R⁴ together with the nitrogen atom to which they are attachedform a moiety selected from: (i) a moiety of formula (a):

(ii) a moiety of formula (b):

(iii) a moiety of formula (c):

wherein: R⁵ is —OC(O)NR^(a)R^(b), —C(O)NR^(a)R^(b),—NR^(d)S(O)₂C₁₋₃alkyl, —NR^(d)C(O)R^(c), NR^(d)S(O)₂NR^(a)R^(b), or—NR^(d)C(O)OR^(e); R⁶ is —C(O)R^(f), —(CH₂)₂OR^(g), —S(O)₂NR^(a)R^(b),—S(O)₂C₁₋₃alkyl, or —S(O)₂(CH₂)₁₋₃S(O)₂C₁₋₃alkyl; R^(a), R^(b), andR^(c) are independently hydrogen or C₁₋₃alkyl; R^(d) is hydrogen orC₁₋₃alkyl, wherein C₁₋₃alkyl is optionally substituted with hydroxy;R^(e) is C₁₋₃alkyl; R^(f) is hydrogen, C₁₋₃alkyl, tetrahydrofuranyl, or—NR^(a)R^(b); R^(g) is hydrogen or C₁₋₃alkyl; a is 0, 1 or 2; b is 0, 1,2 or 3; c is 0, 1, or 2; d is 1 or 2; and e is 1 or 2; provided thatwhen c is 0, then d is 2, and R⁵ is —C(O)NR^(a)R^(b); and when c is 2,then d is 1; or a pharmaceutically-acceptable salt or stereoisomerthereof.
 22. The method of claim 21, wherein the disorder of reducedmotility is chronic constipation, constipation-predominant irritablebowel syndrome, diabetic and idiopathic gastroparesis, or functionaldyspepsia.
 23. The method of claim 21 or 22, wherein, in the compound offormula (I): R² is ethyl or isopropyl R³ is C₁₋₃alkyl; and R⁴ is—(CH₂)₁₋₃C(O)NR^(a)R^(b) or

or R³ and R⁴ together with the nitrogen atom to which they are attachedform a moiety selected from a moiety of formula (a), a moiety of formula(b), and a moiety of formula (c); wherein R⁵ is —OC(O)NR^(a)R^(b) or—C(O)NR^(a)R^(b); R⁶ is —C(O)R^(f), —(CH₂)₂OR^(g), or —S(O)₂NR^(a)R^(b);R^(a), R^(b), and R^(g) are independently hydrogen or methyl; R^(f) ismethyl, tetrahydrofuranyl, or —NR^(a)R^(b); a is 0; b is 1; c is 1 or 2;d is 1; and e is
 1. 24. The method of claim 23, wherein R³ and R⁴together with the nitrogen atom to which they are attached form a moietyof formula (b), wherein R⁶ is —C(O)R^(f).
 25. The method of claim 21 or22, wherein the compound of formula (I) is selected from:4-(tetrahydrofuran-2-carbonyl)piperazine-1-carboxylic acid3-{(1S,3R,5R)-3-[(3-isopropyl-2-oxo-2,3-dihydrobenzoimidazole-1-carbonyl)amino]-8-azabicyclo-[3.2.1]oct-8-yl}propylester; 4-(2-hydroxyethyl)piperazine-1-carboxylic acid3-{(1S,3R,5R)-3-[(3-isopropyl-2-oxo-2,3-dihydrobenzoimidazole-1-carbonyl)amino]-8-azabicyclo-[3.2.1]oct-8-yl}propylester; 4-acetyl-piperazine-1-carboxylic acid3-{(1S,3R,5R)-3-[(3-isopropyl-2-oxo-2,3-dihydro-benzoimidazole-1-carbonyl)amino]-8-azabicyclo[3.2.1]oct-8-yl}propylester; 4-dimethylcarbamoyloxypiperidine-1-carboxylic acid3-{(1S,3R,5R)-3-[(3-isopropyl-2-oxo-2,3-dihydrobenzoimidazole-1-carbonyl)amino]-8-aza-bicyclo[3.2.1]oct-8-yl}propylester; 3-carbamoylpiperidine-1-carboxylic acid3-{(1S,3R,5R)-3-[(3-isopropyl-2-oxo-2,3-dihydro-benzoimidazole-1-carbonyl)amino]-8-azabicyclo[3.2.1]oct-8-yl}propylester; 1,1-dioxo-1λ⁶-thiomorpholine-4-carboxylic acid3-{(1S,3R,5R)-3-[(3-isopropyl-2-oxo-2,3-dihydrobenzoimidazole-1-carbonyl)amino]-8-azabicyclo[3.2.1]oct-8-yl}propylester; (1,1-dioxotetrahydro-1λ⁶-thiophen-3-yl)methylcarbamic acid3-{(1S,3R,5R)-3-[(3-isopropyl-2-oxo-2,3-dihydrobenzoimidazole-1-carbonyl)amino]-8-azabicyclo-[3.2.1]oct-8-yl}propylester; (R)-2-carbamoylpyrrolidine-1-carboxylic acid3-{(1S,3R,5R)-3-[(3-isopropyl-2-oxo-2,3-dihydrobenzoimidazole-1-carbonyl)amino]-8-azabicyclo[3.2.1]oct-8-yl}propylester; 4-acetyl-[1,4]diazepane-1-carboxylic acid3-{(1S,3R,5R)-3-[(3-isopropyl-2-oxo-2,3-dihydro-benzoimidazole-1-carbonyl)amino]-8-azabicyclo[3.2.1]oct-8-yl}propylester; dimethylcarbamoylmethyl-methylcarbamic acid3-{(1S,3R,5R)-3-[(3-isopropyl-2-oxo-2,3-dihydrobenzoimidazole-1-carbonyl)amino]-8-azabicyclo[3.2.1]oct-8-yl}propylester; 4-dimethylcarbamoylpiperazine-1-carboxylic acid3-{(1S,3R,5R)-3-[(3-isopropyl-2-oxo-2,3-dihydrobenzoimidazole-1-carbonyl)amino]-8-azabicyclo[3.2.1]oct-8-yl}propylester; and 4-dimethylsulfamoylpiperazine-1-carboxylic acid3-{(1S,3R,5R)-3-[(3-isopropyl-2-oxo-2,3-dihydrobenzoimidazole-1-carbonyl)amino]-8-azabicyclo[3.2.1]oct-8-yl}propylester; and pharmaceutically-acceptable salts thereof.
 26. The method ofclaim 21 or 22 wherein the compound of formula (I) is1,1-dioxo-1λ⁶-thiomorpholine-4-carboxylic acid3-{(1S,3R,5R)-3-[(3-isopropyl-2-oxo-2,3-dihydrobenzoimidazole-1-carbonyl)amino]-8-azabicyclo[3.2.1]oct-8-yl}propylester or a pharmaceutically-acceptable salt thereof.
 27. A method oftreating a mammal having a medical condition ameliorated by treatmentwith a 5-HT₄ receptor agonist, the method comprising administering tothe mammal, a therapeutically effective amount of a pharmaceuticalcomposition comprising a pharmaceutically-acceptable carrier and acompound of formula (I) as defined in claim 21 or apharmaceutically-acceptable salt of stereoisomer thereof.
 28. The methodof claim 27 wherein the medical condition is irritable bowel syndrome,chronic constipation, functional dyspepsia, delayed gastric emptying,gastroesophageal reflux disease, gastroparesis, post-operative ileus,intestinal pseudo-obstruction, or drug-induced delayed transit.